Reagents and Methods for Esterification

ABSTRACT

Methods and reagents for esterification of biological molecules including proteins, polypeptides and peptides. Diazo compounds of formula I: 
     
       
         
         
             
             
         
       
     
     where R is hydrogen, an alkyl, an alkenyl or an alkynyl, R A  represents 1-5 substituents on the indicated phenyl ring and R M  is an organic group. R M  includes a label, a cell penetrating group, a cell targeting group, or a reactive group or latent reactive group for reaction to bond to a label, a cell penetrating group, or a cell targeting group, among other organic groups useful for esterification of biological molecules. Also provided are diazo compounds which are bifunctional and trifunctional coupling reagents as well as reagents for the synthesis of compounds of formula I.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 15/730,197,filed Oct. 11, 2017, now allowed, which in turn is a division of U.S.application Ser. No. 15/093,510, filed Apr. 7, 2016, now U.S. Pat. No.9,790,483, issued Oct. 17, 2017, which claims the benefit of U.S.Provisional Application 62/145,193, filed Apr. 9, 2015, and U.S.Provisional Application 62/319,153, filed Apr. 6, 2016. Each of thelisted applications is incorporated by reference herein in its entirety.

STATEMENT REGARDING GOVERNMENT SUPPORT

This invention was made with government support under GM044783 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

Chemoselective transformations [1-3] are of key importance in modernchemical biology. Proteins, peptides and amino acids have carboxylgroups in side chains and at the C-terminus. Methods and reagents forselective esterification of such carboxyl groups, particularly those inpolypeptides and proteins, which are efficient and give high yield andwhich can be carried out in buffered aqueous solution are of particularinterest. Esterification reactions that do not require a catalyst arealso of particular interest. Protein esterification can for example beemployed for protein labeling (isotopic, radiolabeling, or fluorescentlabeling) and to provide a way to controllably and efficiently increaseprotein lipophilicity or increase the positive charge on the protein andtherefore promote cellular uptake. [20]

It is also of interest for certain applications that the esters formedare “bio-reversible” such that the ester groups are removable byesterases. In a specific application, esterification can be employed tofunctionalize a protein with moieties that direct the protein towards aparticular cell type or and/or which facilitate its cellular uptake. Ifesterification is bio-reversible, the groups added to target the proteinto a cell or to enhance its uptake into the cell can be removed byendogenous enzymes in the cell to regenerate native protein.

Diazo groups are one of the most versatile functional groups insynthetic organic chemistry. [23a-e, 4, 24] It has recently beenreported that diazo-compounds can be employed in place of azides as the1,3-dipole in 1,3-dipolar cycloaddition reactions with alkynes. [4] Therates can greatly exceed those of the analogous azide [4] and thereactions are chemoselective in the presence of mammalian cells. [24]The use of diazo-com pounds in such reactions was at least in part madefeasible with the availability of methods that convert azides intodiazo-compounds using a phosphinoester. [5] These methods are describedin U.S. Pat. No. 8,350,014 which is incorporated by reference herein inits entirety for its description of such methods and diazo-compoundsprepared by the methods. In addition, diazo compounds have been used tolabel proteins via C—H and N—H insertion reactions. [25a,b]

The esterification of carboxylic acids with diazomethane has biologicalpotential, but suffers from non-specific reactivity with the hydroxylgroups tyrosine side chains and the amino groups on lysine side chains.[6] In addition, this process only provides access to methyl esters,which are not particularly useful in biologic systems due to theirnon-specific lability toward various esterases present in biologicalmilieu. [7] Compounds with targeted specificity for common biologicfunctional moieties that preclude deleterious side reactions areparticularly useful. [8]

Stabilized diazo compounds have found widespread use in syntheticorganic chemistry. [9] This is primarily due to their ability to reactwith carboxylic acids and amides by forming metal carbenoids [10] tofacilitate O—H or N—H bond insertion respectively. [11,12] In an effortto avoid the use of toxic metals, it was reported that fluorous organicsolvents [13] were sufficient to help facilitate the reaction due totheir high polarity and poor nucleophilicity. [14] Additionally, variousnon-stabilized diazo compounds generated in situ were shown to becapable of carrying out the esterification of carboxylic acids [15], buttheir unstable nature limits their biological utility.

Early use of stabilized diazo compounds in a biological context involvedadding diazo glycinamide [16], diphenyldiazomethane [17] ordiazoacetamide [18,19] to identify the reactive carboxylic acids onproteins. These methods all required adding a vast excess of the diazocompound and tedious monitoring of reaction pH to achieve modestlabeling. Moreover, the reaction was not chemoselective, as amino,sulfhydryl, and phenolic side chains suffered alkylation. Suchmodifications are potentially deleterious to protein function and notbioreversible. [30]

It has recently been reported that the basicity of 9-diazofluoreneendows this diazo compound with the ability to label a carboxyl group ofa protein in an aqueous environment. [4] A comparison of the reactivityof 9-diazofluorene with that of N-benzyl-2-diazoacetamide with variouscarboxylic acids in acetonitrile and acetonitrile/aqueous buffer(3:1v/v) demonstrated that while both diazo compounds gave the desiredesters in the organic solvent, only 9-diazofluorene gave the desiredester in aqueous medium. In contrast, diethyl 2-diazomalonate was foundto be unreactive for ester formation in the organic or aqueous medium.Reactivity of the diazo compound to form the desired esters in aqueousmedium was reported to be associated with the ability of the diazocompound to abstract a proton from a carboxylic acid. Further, thisability to abstract a proton was reported to be associated with thepK_(a) (as measured in dimethylsulfoxide) [21] of the conjugate acid ofthe organic moiety bonded to the diazo group (e.g., conjugate acids ofdiethylmalonate (pK_(a)=16.4), fluorene (pK_(a)=22.6) anddiethylacetamide (pK_(a) 35). 9-Diazofluorene was reported to function(at 10 eq) to label on average three of eleven carboxylates in RNase A.

While there has been some success in the development of reagents andmethods for the chemoselective generation of biological esters fromcarboxylic acids for protein labeling and other useful proteinmodification, there remains a need in the art for more efficientchemoselective esterification reagents for proteins and other biologicalentities (e.g., nucleic acids) which result in bioreversible esterformation. Additionally, there remains a need in the art forchemoselective esterification reagents that are synthetically amenableto modification with biologically useful entities.

SUMMARY OF THE INVENTION

The invention provides methods and reagents for esterification ofbiological molecules including proteins, polypeptides and peptides. Theinvention provides certain diazo compounds of formula I:

where R_(A) represents 1-5 substituents on the indicated phenyl ring, Ris hydrogen, an alkyl, alkenyl or alkynyl group, and R_(M) is an organicgroup, which can include a label, a cell penetrating group, a celltargeting group, or a reactive group or latent reactive group forreaction to bond to a label, a cell penetrating group, or a celltargeting group, among other organic groups. R_(M) optionally includes aspacer or linker group. In a specific embodiment, the cell targetinggroup is a protein, a polypeptide or a peptide. In a specificembodiment, the cell targeting group is an antibody or functionalfragment thereof. Diazo compounds of formula I are useful to convertcarboxylic acid groups of biological molecules, particularly those ofthe side chains and C-terminus of proteins, polypeptides and peptidesinto esters, by reaction of the diazo group. In specific embodiments,the esterification is carried out in buffered aqueous solvent at pHranging from 5-7 and preferably at pH ranging from 5.5 to 6.5 and doesnot require the use of a catalyst. In specific embodiments, R_(M) is anoptionally substituted alkyl, alkenyl, alkynyl or aryl group. Inspecific embodiments, R and R_(M), together with the nitrogen to whichthey are bonded, form an optionally substituted 5- to 10-member ringsystem, which optionally contains one or two heteroatoms in addition tothe N. In specific embodiments, R_(M) is an optionally substitutedalkyl, alkenyl or alkynyl group having 1, 2, 3, 4, 5 or 6 carbon atoms.In specific embodiments, R is hydrogen, methyl or ethyl. In specificembodiments, R is hydrogen, methyl, or ethyl and R_(M) is an optionallysubstituted alkyl, alkenyl or alkynyl group having 1, 2, 3, 4, 5 or 6carbon atoms. In specific embodiments, R is hydrogen, methyl, or ethyland RM is an alkyl, alkenyl or alkynyl group having 1, 2, 3, 4, 5 or 6carbon atoms. Optional substitution of alkyl, alkenyl, or alkynyl groupsincludes substitution with non-hydrogen substituents selected fromalkyl, alkoxy, halogen, haloalkyl or haloalkoxy.

In a specific embodiment, diazo-compounds are those of formula I where:R is hydrogen, an optionally substituted alkyl, alkenyl or alkynylgroup; R_(A) represents hydrogens at each phenyl ring position, orrepresents 1 to 5 non-hydrogen substituents on the phenyl ring (anyremaining ring positions carrying hydrogens), wherein the non-hydrogensubstituents are selected from the group consisting of alkyl,cycloalkyl, alkoxy, cycloalkoxy, alkenyl, alkenyloxy, aryl, aryl oxy,arylalkyl, arylalkyloxy, halogen, haloalkyl, haloalkoxy, heterocyclyl,sulfhydryl (—SH), thioalky (—S-alkyl), —NH₂ and —NH—CO—R_(P), where thealkyl, cycloalkyl, alkoxy, cycloalkoxy, alkenyl, alkenyloxy, aryl,aryloxy, arylalkyl, arylalkyloxyand heterocyclyl groups are optionallysubstituted with 1-3 non-hydrogen substituents selected from alkyl,alkoxy, halogen, haloalkyl or haloalkoxy groups and R_(P) is hydrogen,an alkyl group or R_(M1); and

-   -   R_(M) or R_(M1) are independently an optionally substituted        organic group M or M₁, respectively, having from 1 to 100 carbon        atoms and optionally nitrogen, oxygen or sulfur atoms, or —L-M,        or —L₁-M₁, respectively, where —L— and —L₁— are independently a        divalent linker moiety having from 1-30 carbon atoms and        optionally nitrogen, oxygen or sulfur atoms; or    -   R_(M) or R_(M1) is or comprises a polymer, such as polyethylene        glycol, where the polymer is directly bonded into the compound        or is bonded via a linker (—L— and —L₁—).

In specific embodiments, R_(M) or R_(M1) is a cargo molecule. In aspecific embodiment, both of R_(M) or R_(M1) are cargo molecules. Inspecific embodiments, R_(M) or R_(M1) is or comprises a polymer. Inspecific embodiments, R_(M) or R_(M1) is or comprises a hydrophilicpolymer. In specific embodiments, R_(M) or R_(M1) is or comprises ahydrophilic polymer having number average molecular weight of 10,000 orless. In specific embodiments, the polymer. is polyethylene glycol. Inspecific embodiments, the polyethylene glycol has number averagemolecular weight less than 10,000. In specific embodiments, R_(A)represents 1 to 3 non-hydrogen substituents on the phenyl ring (anyremaining ring positions carrying hydrogens). In specific embodiments,R_(A) represents 1 or 2 non-hydrogen substituents on the phenyl ring(any remaining ring positions carrying hydrogens). In specificembodiments, R_(A) represents 1 non-hydrogen substituents on the phenylring (any remaining ring positions carrying hydrogens).

The invention also provides a method for esterifying one or morecarboxylic acid groups in an organic or biological molecule whichcomprises contacting the organic or biological molecule with adiazo-compound of formula I. Esterification employing diazo-compounds offormula I can, dependent upon R_(M) and/or R_(M1), facilitate labeling,cell targeting, and/or cell penetration of the species (e.g., a protein)which is esterified. Compounds of formula I where R_(M) and/or R_(M1) isor comprises a reactive group or a latent reactive group can be employedas bifunctional or trifunctional reagents to bond other R_(M) and/orR_(M1) groups which are, for example, labels, cell penetrating groups,or cell targeting groups to the diazo-moiety of formula I. Morespecifically, compounds of formula I where R_(M) and/or R_(M1) is orcomprises a reactive group or a latent reactive group can be employed asheterobifunctional or heterotrifunctional reagents where reactive andlatent reactive groups have orthogonal reactivity.

In specific embodiments, the invention provides compounds of formula II

where R_(A) is defined as for formula I above and wherein AC representsthe leaving group of an activated ester. Compounds of formula V areuseful at least as reagents in the preparation of the compounds offormula I. Activated esters include among others, N-hydroxysuccinimideesters (NHS esters), N-hydroxysulfosuccinimide esters (sulfo-NHSesters), N-hydroxyphthalimide esters, phenyl esters where the phenylgroup is substituted with one or more electron withdrawing groups (e.g.,nitro groups or halogens), optionally substituted alkyl or arylsulfonate esters (e.g., tosyl esters, mesyl esters, or triflate esters).These compounds are useful, at least, for the preparation of diazoesterification reagents of this invention.

In specific embodiments of formula II, R_(A) represents substitution ofthe indicated ring with 1 to 5 non-hydrogen substituents on the phenylring (any remaining ring positions carrying hydrogens), wherein thenon-hydrogen substituents are selected from the group consisting ofalkyl, cycloalkyl, alkoxy, cycloalkoxy, alkenyl, alkenyloxy, aryl, aryloxy, arylalkyl, arylalkyl oxy, halogen, haloalkyl, haloalkoxy,heterocyclyl, sulfhydryl (—SH), thioalky (—S-alkyl), —NH₂ and—NH—CO—R_(P), where the alkyl, cycloalkyl, alkoxy, cycloalkoxy, alkenyl,alkenyloxy, aryl, aryloxy, arylalkyl, arylalkyloxy and heterocyclylgroups are optionally substituted with 1 to 5 non-hydrogen substituentsselected from alkyl, alkoxy, halogen, haloalkyl or haloalkoxy groups andR_(P) is hydrogen, an alkyl group or R_(M1), where R_(M1) is as definedfor formula I. In specific embodiments of formula II, R_(A) is in thepara position on the phenyl ring. In specific embodiments, R_(A) isp-alkyl. In specific embodiments, R_(A) is p-methyl. In specificembodiments, R_(A) is p-alkyloxy. In specific embodiments, R_(A) isp-methoxy.

In specific embodiments, the invention provides reagents of formula IIA:

where R_(A) is as defined for formula I, and E is hydrogen or —SO₃ ⁻(sulfo) salt (e.g., a sodium salt). In specific embodiments of formulaIIA, R_(A) is in the para position on the phenyl ring. In specificembodiments, R_(A) is p-alkyl. In specific embodiments, R_(A) isp-methyl. In specific embodiments, R_(A) is p-alkyloxy. In specificembodiments, R_(A) is p-methoxy.

Additional aspects and embodiments of the invention will become apparentto one of ordinary skill in the art on review of the following detaileddescription and non-limiting examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates a scaffold for testing the reactivity andselectivity of diazo compounds. FIG. 1B illustrates the synthetic routeto diazo compounds 1-6 where the steps are a) NBS, AIBN; b) NaN₃,THF:H₂O; c) NHS, DCC, THF; d) PhCH₂NH₂, DCM; e) N-succinimidyl3-(diphenylphosphino)propionate, then NaHCO₃ or DBU [5a,b], f)imidazole-1-sulfonyl azide hydrochloride, DBU, CuSO₄, MeOH [29].

FIG. 2A shows a table of second-order rate constants for theesterification of BocGlyOH by diazo compounds 1-6 in CD₃CN. FIG. 2Billustrates a Hammett plot of the data in panel A. Values of σ_(p) arefrom Hansch et al. [32]. ρ=−2.7.

FIG. 3A illustrates the reaction to form ester or alcohol. FIG. 3Bprovides a graph showing the effect of σ_(p) value on thechemoselectivity (ester/alcohol product ratio) of diazo compounds 1-6 in1:1 buffer:acetonitrile at the bottom of the figure.

FIG. 4 illustrates the reaction of diazo-compound 2 to form ester oralcohol products with acids a-c at the top of the figure. FIG. 4 alsoshows structures of acids a-c and provides a Table of product ratiosillustrating chemoselectivity of esterification reactions ofdiazo-compound 2 in aqueous solution at the bottom of the figure.

FIGS. 5A-B provide MALDI-TOF mass spectrometry data for esterificationof RNase A with 9-diazofluorene and diazo compound 2, respectively.

FIG. 6A Illustrates the ultraviolet spectra of diazo compound 2 measuredover the concentration range 0.8-50 mM. FIG. 6B provides a plot of theconcentration dependence of the absorbance of diazo compound 2 (0.8-50mM) at λmax=435 nm which gave ϵ=30.5 M-1 cm-1.

FIGS. 7A-B report quantification of labeling efficiency of GFP bycertain diazo compounds. FIG. 7A shows the compound structure and log Pfor certain diazo compounds. FIG. 7B is a graph showing the number oflabels added to GFP for each diazo compound as determined with MALDI-TOFmass spectrometry.

FIG. 8 is a graph showing quantification of internalization of labeledversus unlabeled GFP by CHO K1 cells using flow cytometry measuringmedian fluorescence intensity. Single cells were sorted based on forwardscatter-side scatter measurements, and live cells were sorted using 7AAD(7-amino-actinomycin D) stain.

FIGS. 9A-C illustrate microscopy images of uptake of esterified GFP(green) by CHO K1 cells. FIG. 9A: Not esterified. FIG. 9B: Esterifiedwith α-diazo-4-methylphenyl-N-propargylacetamide. FIG. 9C: Esterifiedwith α-diazo-4-methylphenyl-N,N-dimethylacetamide. Cell nuclei arestained with Hoechst 33342 (blue).

DETAILED DESCRIPTION OF THE INVENTION

This invention is based at least in part on studies of the reactivity ofcertain diazo-compounds for esterification of carboxylic acid groups asa function of their structure and electronic properties. Diazo compoundscan function for esterification of carboxylic acids. This reactivity canprovide unique opportunities in chemical biology. For example, unlikethe alkylation of other functional groups, O-alkylation of a carboxylgroup is bioreversible because mammalian cells contain non-specificesterases.[7, 26 a-c]. The esterification of carboxyl groups in proteinsand other biomolecules is, however, difficult to effect, as solventwater competes effectively with alcohols for eletrophilic acyl groups.In contrast, esterification reactions mediated by diazo groups rely onthe carboxyl group serving as a nucleophile (Scheme 1). [27a,b].

Attempts have been made to use diazo compounds to label proteins. [19,28a-c] A large molar excess (up to 103-fold) of diazo compound wasrequired to overcome hydrolytic decomposition. Moreover, the reactionwas not chemoselective, as amino, sulfhydryl, and phenolic side chainssuffered alkylation. Such modifications are potentially deleterious toprotein function and not bioreversible. [30].This invention relates todiazo-com pounds exhibiting improved esterification of carboxyl groupsin an aqueous environment. Derivatives of phenylglycinamide (see FIG.1A) have been investigated. This scaffold delocalizes the electrondensity on Ca into an amidic carbonyl group as well as a phenyl groupthat enables a Hammett analysis [31a-d] of the esterification reaction.

The invention provides methods and reagents for esterification ofbiological molecules including proteins, polypeptides and peptides. Theinvention provides certain diazo compounds of formula I:

where R_(A) represents 1-5 substituents on the indicated phenyl ring, Ris hydrogen, an alkyl, alkenyl or alkynyl group, and R_(M) is generallyan organic group, which can includes a label, a cell penetrating group,a cell targeting group, or a reactive group or latent reactive group forreaction to bond to a label, a cell penetrating group, or a celltargeting group, among other organic groups. R_(M) optionally includes aspacer or linker group. In a specific embodiment, the cell targetinggroup is a protein, a polypeptide or a peptide. In a specificembodiment, the cell targeting group is an antibody or functionalfragment thereof. Diazo compounds of formula I are useful to convertcarboxylic acid groups of biological molecules, particularly those ofthe side chains and C-terminus of proteins, polypeptides and peptidesinto esters, by reaction of the diazo group. In specific embodiments,the esterification is carried out in buffered aqueous solvent at pHranging from 5-7 and preferably at pH ranging from 5.5 to 6.5 and doesnot require the use of a catalyst. In specific embodiments, R_(M) is anoptionally substituted alkyl, alkenyl, alkynyl or aryl group. Inspecific embodiments, R and R_(M) together with the nitrogen to whichthey are bonded form an optionally substituted 5 to 10 member ringsystem, which optionally contains one or two heteroatoms in addition tothe N. In specific embodiments, R_(M) is an optionally substitutedalkyl, alkenyl or alkynyl group having 1, 2, 3, 4, 5 or 6 carbon atoms.In specific embodiments, R is hydrogen, methyl or ethyl. In specificembodiments, R is hydrogen, methyl, or ethyl and R_(M) is an optionallysubstituted alkyl, alkenyl or alkynyl group having 1, 2, 3, 4, 5 or 6carbon atoms. In specific embodiments, R is hydrogen, methyl, or ethyland R_(M) is an alkyl, alkenyl or alkynyl group having 1, 2, 3, 4, 5 or6 carbon atoms. Optional substitution of alkyl, alkenyl, or alkynylgroups includes substitution with non-hydrogen substituents selectedfrom alkyl, alkoxy, halogen, haloalkyl or haloalkoxy.

In a specific embodiment, diazo-compounds useful in the invention arethose of formula I where:

-   -   R is hydrogen, an optionally substituted alkyl, alkenyl or        alkynyl group;    -   R_(A) represents hydrogens at each phenyl ring position, or        represents 1 to 5 non-hydrogen substituents on the phenyl ring        (any remaining ring positions carrying hydrogens), wherein the        non-hydrogen substituents are selected from the group consisting        of alkyl, cycloalkyl, alkoxy, cycloalkoxy, alkenyl, alkenyloxy,        aryl, aryl oxy, arylalkyl, arylalkyl oxy, halogen, haloalkyl,        haloalkoxy, heterocyclyl, sulfhydryl (—SH), thioalky (—S-alkyl),        —NH₂ and —NH—CO—R_(P), where the alkyl, cycloalkyl, alkoxy,        cycloalkoxy, alkenyl, alkenyloxy, aryl, aryloxy, arylalkyl,        arylalkyloxyand heterocyclyl groups are optionally substituted        with 1-3 non-hydrogen substituents selected from alkyl, alkoxy,        halogen, haloalkyl or haloalkoxy groups and R_(P) is hydrogen,        an alkyl group or R_(M1); and    -   R_(M) or R_(M1) are independently an optionally substituted        non-polymeric organic group M or M₁, respectively, having from 1        to 100 carbon atoms and optionally nitrogen, oxygen or sulfur        atoms, or —L—M, or —L₁—M₁, respectively, where —L— and —L₁— are        independently a divalent linker moiety having from 1-30 carbon        atoms and optionally nitrogen, oxygen or sulfur atoms; or    -   R_(M) or R_(M1) is or comprises a polymer, such as polyethylene        glycol where the polymer is directly bonded into the compound or        is bonded via a linker (—L— and —L₁—).

In specific embodiments, R_(M) or R_(M1) is a cargo molecule. In aspecific embodiment, both of R_(M) or R_(M1) are cargo molecules. Inspecific embodiments, R_(M) or R_(M1) is or comprises a polymer. Inspecific embodiments, R_(M) or R_(M1) is or comprises a hydrophilicpolymer. In specific embodiments, R_(M) or R_(M1) is or comprises ahydrophilic polymer having number average molecular weight of 10,000 orless. In specific embodiments, the polymer. is polyethylene glycol. Inspecific embodiments, the polyethylene glycol has number averagemolecular weight less than 10,000.

In specific embodiments, R_(A) represents 1 to 3 non-hydrogensubstituents on the phenyl ring (any remaining ring positions carryinghydrogens). In specific embodiments, R_(A) represents 1 or 2non-hydrogen substituents on the phenyl ring (any remaining ringpositions carrying hydrogens). In specific embodiments, R_(A) representsone non-hydrogen substituents on the phenyl ring (any remaining ringpositions carrying hydrogens).

In specific embodiments, R_(A) represents ring substitution having atleast one non-hydrogen group as listed above at the para ring position.In specific embodiments, R_(A) represents ring substitution having atleast one non-hydrogen group as listed above at a meta ring position.

The compound of formula I optionally has one or two sites for furtherfunctionalization through the R_(P)—CO—NH— group on the phenyl ring(left in the above structure) or through R_(M) on the phenyl ring (rightin the above structure).

In specific embodiments, R_(A) includes ring substitution with a groupwhich has a Hammett σ_(p) (para-sigma) or σ_(m) (meta-sigma) value of−0.2 to +0.1. In specific embodiments, R_(A) includes ring substitutionwith a group which has a Hammett a (para-sigma) or σ_(m) (meta-sigma)value of −0.17 to +0.1. In specific embodiments, R_(A) includes ringsubstitution with a group which has a Hammett σ_(p) (para-sigma) orσ_(m) (meta-sigma) value of −0.17 to +0.05. In specific embodiments,R_(A) includes ring substitution with a group which has a Hammett σ_(p)(para-sigma) or σ_(m) (meta-sigma) value of −0.17 to 0.

In specific embodiments, R_(A) is substitution with a group which has aHammett σ_(p) (para-sigma) or σ_(m) (meta-sigma) value of −0.2 to +0.1.In specific embodiments, R_(A) is substitution with a group which has aHammett σ_(p) (para-sigma) or σ_(m) (meta-sigma) value of −0.17 to +0.1.In specific embodiments, R_(A) is substitution with a group which has aHammett σ_(p) (para-sigma) or σ_(m) (meta-sigma) value of −0.17 to+0.05. In specific embodiments, R_(A) is substitution with a group whichhas a Hammett σ_(p) (para-sigma) or σ_(m) (meta-sigma) value of −0.17 to0.

In specific embodiments, R_(A) is substitution at the para, meta or bothring position with a group which has a Hammett σ_(p) (para-sigma) orσ_(m) (meta-sigma) value of -0.2 to +0.1. In specific embodiments, R_(A)is substitution at the para, meta or both ring position with a groupwhich has a Hammett σ_(p) (para-sigma) or σ_(m) (meta-sigma) value of-0.17 to +0.1. In specific embodiments, R_(A) is substitution at thepara, meta or both ring position with a group which has a Hammett σ_(p)(para-sigma) or σ_(m) (meta-sigma) value of -0.17 to +0.05. In specificembodiments, R_(A) is substitution at the para, meta or both ringposition with a group which has a Hammett σ_(p) (para-sigma) or σ_(m)(meta-sigma) value of −0.17 to 0.

In specific embodiments, R_(A) represents substitution with one or moresubstituents selected from alkyl groups having one to four carbon atoms,an alkenyl group having one double bond and two to four carbon atoms, anunsubstituted phenyl group, a alkylthio group (—S-alkyl) having one tofour carbon atoms, an —NHCOR_(N) group where R_(N) is H or a methylgroup, fluorine, or —NH₂. In specific embodiments, R_(A) representssubstitution at the para position on the ring with an alkyl group having1-4 carbon atoms, an alkenyl group having one double bond and one tofour carbon atoms, a fluorine, an alkylthio group, a phenyl group, a—NHCOH group or a —NHCOCH₃ group. In specific embodiments, R_(A)represents substitution at the meta position on the ring with an alkylgroup having 1-4 carbon atoms, a phenyl group, or an —NH₂ group.

In a specific embodiment R_(A) is a single group in the para position onthe phenyl ring of formula I. In a specific embodiment R_(A) is a singlegroup in a meta position on the phenyl ring of formula I.

In specific embodiments, R_(A) is an alkyl group having 1-6 carbonatoms. In more specific embodiments, R_(A) is methyl or ethyl. Inspecific embodiments, R_(A) is a single alkyl group having 1-6 carbonatoms in the para position on the phenyl ring of formula I. In specificembodiments, R_(A) is a single methyl or ethyl group in the paraposition on the phenyl ring of formula I. In specific embodiments, R_(A)is a methyl group in the para position on the phenyl ring of formula I.

In specific embodiments, R_(A) is a heterocyclyl group bonded to thephenyl ring of formula I via a nitrogen. In specific embodiments, R_(A)is a heterocyclyl group bonded to the para position of the phenyl ringof formula I via a nitrogen. In specific embodiments, R_(A) is a singlepiperazinyl group or a morpholino group bonded to the phenyl ring viathe ring N of the group. In specific embodiments, R_(A) is a singlepiperazinyl group or a morpholino group bonded to the para position ofthe phenyl ring via the ring N of the group.

In a specific embodiment, R_(A) is a R_(P)—CO—NH— group where is R_(P)is R_(M1) or M1. In specific embodiments, a single R_(P)—CO—NH— group isbonded at the para position of the phenyl ring in formula I.

In embodiments, R_(M) and R_(M1) are independently an organic grouphaving from 1-20, 1-30, 1-40 or 1-50 carbon atoms and optionally havingnitrogen, oxygen or sulfur atoms. In specific embodiments, R_(M) and/orR_(M1) has 1, 2, 3, 4, or 6 heteroatoms selected from nitrogen, oxygenor sulfur. In embodiments, —L— and/or —L₁— is a linker moiety havingfrom 1-6, 1-10 or 1-20 carbon atoms and optionally nitrogen, oxygen orsulfur atoms. In embodiments, —L— and/or —L₁— has 1-4, oxygen atoms(—O—). In embodiments, —L— and/or -Li- has 1-4 —CO— moieties. Inembodiments, —L— and/or —L₁— has 1-4 -N—R_(N)— moieties, where —R_(N) ishydrogen or an alkyl group having 1-3 carbon atoms. In embodiments, —L—and/or —L₁— has 1-4 —S— moieties. In embodiments, —L— and/or —L₁— hasone —S—S— moiety. In embodiments, —L— and/or —L₁— has 1 or 2 —CO—,—NR_(N)— or —NR_(N)—CO— moieties. In embodiments, —L— and/or —L₁— has 1or 2 —CO— or —O—CO-moieties.

In specific embodiments, —L— and/or —L₁— are or comprise an alkenylenemoiety —(CH₂)q—, where q is an integer from 1 to 6, 1-12 or 1-20. Inspecific embodiments, —L— and/or —L₁— are or comprise an alkoxyalkyl orether group, e.g., —[O]a-[(CH₂)b-O—]r-(CH₂)c-, where a is 0 or 1, b andc are independently an integer from 0 to 6 (where one of b or c is not0), and r is 0 or is an integer from 1-3, 1-6 or 1-10. In specificembodiments, —L— and/or —L₁— are or comprise an alkoxyalkyl or ethergroup, e.g., —[N-R_(N)]d-[(CH₂)b-O—]r-(CH₂)c-, where d is 1, b and c areindependently an integer from 0 to 6 (where one of b or c is not 0), andr is 0 or is an integer from 1-3, 1-6 or 1-10. In specific embodiments,R_(N) is hydrogen, b is 2 or 3, r is 1-3 or 1-6 and c is 0 or 1. Inspecific embodiments, —L— and/or -Li- are or comprise amino moieties,e.g., -[NR_(N)]a-[(CH₂)b-NR_(N)-]r-(CH₂)c-, where a is 0 or 1, b and care independently an integer from 0 to 6 (where one of b or c is not 0),and r is 0 or is an integer from 1-3, 1-6 or 1-10. In specificembodiments, each R_(N) is hydrogen, b is 2 or 3, r is 1-3 or 1-6 and cis 0, 2 or 3. In specific embodiments, —L— and/or —L₁— comprise one ortwo X moieties at either end of the moiety which function for linkage ofthe spacer, where X is selected from —CO—, —OCO—, —CO—NR_(N)—, or—R_(N)—CO—.

In an embodiment, R_(M) is an alkyl, cycloalkyl, aryl, arylalkyl,heterocyclyl or heteroaryl group which is optionally substituted withone or more alkyl, alkoxy, aryl, alkylaryl, halogen, haloalkyl, orhaloalkoxy groups. In an embodiment, R_(M) is an alkyl, cycloalkyl,aryl, arylalkyl, heterocyclyl or heteroaryl group which is optionallysubstituted with one or more alkyl, alkoxy, aryl, alkylaryl, halogen,haloalkyl, or haloalkoxy groups.

In an embodiment, R_(P) is an alkyl, cycloalkyl, aryl, arylalkyl,heterocyclyl or heteroaryl group which is optionally substituted withone or more alkyl, alkoxy, aryl, alkylaryl, halogen, haloalkyl, orhaloalkoxy groups. In an embodiment, R_(P) is an alkyl, cycloalkyl,aryl, arylalkyl, heterocyclyl or heteroaryl group which is optionallysubstituted with one or more alkyl, alkoxy, aryl, alkylaryl, halogen,haloalkyl, or haloalkoxy groups.

In an embodiment, R_(M) or R_(M1) comprise or are independently a labelor reporter molecule (e.g., a fluorescent label, an isotopic label, animaging agent, a quantum dot, and the like). In an embodiment, the labelor reporter is indirectly bonded to the diazo-compound of formula I via—L— or —L₁—. In a specific embodiment, only one of R_(M) or R_(M1) is orcomprises a label or reporter.

In a specific embodiment, R_(M) or R_(M1) is or comprises biotin or aderivative thereof. In a specific embodiment, biotin or a derivativethereof is directed bonded in the compound of formula I or is indirectlybonded therein via a linker.

In an embodiment, R_(M) or R_(M1) comprises or is a cell penetratinggroup, such as a cationic domain, including peptidic cationic species(e.g., HIV-TAT, penetratin, and polyarginine (e.g., nona-arginine) andmore generally cell penetrating peptides (CPP), which are also calledprotein transduction domains (PTDs) or non-peptidic cationic species(e.g., PAMAM dendrimers and polyethylenimine), guanidinium, positivelycharged amines, hydrophobic groups such as fluorenyl or pyrene, whichare optionally bonded via an -alkylene-CO₂- (e.g., pyrenebutyrate),optionally substituted fluorenyl groups or optionally substitutedphenylboronates. In an embodiment, the cell penetrating group isindirectly bonded to the diazo-compound of formula I via —L— or —L₁—. Ina specific embodiment, only one of R_(M) or R_(M1) is or comprises acell penetrating group.

In an embodiment, R_(M) or R_(M1) comprises or is a cell targetinggroup, such as a ligand for a cell-surface receptor (e.g., a steroid,folic acid, substance P, and the RGD tripeptide) or other targetingspecies such as nuclear localization peptides. The targeting groups canbe a protein, polypeptide or peptide. The targeting groups may be anantibody or functional fragment thereof. In an embodiment, the celltargeting group is indirectly bonded to the diazo compound of formula Ivia —L— or —L₁—. In a specific embodiment, only one of R_(M) or R_(M1)is or comprises a cell targeting group.

Exemplary cell targeting groups are described in Srinivasarco et al.[38]. This reference is incorporated by reference herein fordescriptions of ligands for cell targeting. In a specific embodiment,the ligand employed for cell targeting should exhibit an affinity forits receptor of dissociation constant of 10 nM or lower. In a specificembodiment, more than one cell targeting group may be employed in agiven compound of formula I.

In an embodiment, R_(M) or R_(M1) comprises or is a reactive group, suchas a group that reacts with an amine or a thiol. In a specificembodiment, R_(M) is or comprises an amine reactive group, such as anN-hydroxy-succinimide ester group an N-hydroxy-sulfosuccinimide estergroup. In a specific embodiment, R_(M) is or comprises an amine reactivegroup, such as an N-hydroxyphthalimide ester group. In another specificembodiment, R_(M) is an amine reactive activated ester such as ap-nitrophenyl ester group or a pentafluorophenyl ester group. In anotherspecific embodiment, R_(M) is a thiol reactive group, such as a2-pyridyldithio group or an iodoacetyl group. In an embodiment, thefunctional group is bonded indirectly to the diazo-compound of formula Ivia —L— or —L₁—. In a specific embodiment, one of R_(M) or R_(M1) is orcomprises a reactive group, such as an amine reactive group. In aspecific embodiment, both of R_(M) and R_(M1) are or comprise a reactivegroup, particularly where the reactivity of the two reactive groups isorthogonal, such as where one is an amine reactive group and the otheris a thiol reactive group.

In an embodiment, R_(M) or R_(M1) comprises or is a latent reactivegroup which is capable of being activated for reaction with an amine,thiol, alcohol or carboxylate. In an embodiment, R_(M) or R_(M1)comprises or is a latent reactive group carrying a protective groupwhich is selectively removable to activate the latent reactive group forreaction. In an embodiment, the latent reactive group is bondedindirectly to the diazo-compound of formula I via —L— or —L₁—. In aspecific embodiment, one of R_(M) or R_(M1) is or comprises a latentreactive group. In a specific embodiment, both of R_(M) and R_(M1) areor comprise a latent reactive group. In a specific embodiment, one ofR_(M) or R_(M1) is or comprises a reactive group, such as an aminereactive group and the other is a latent reactive group, such as aprotected amine reactive group. In a specific embodiment, both of R_(M)and R_(M1) are or comprise a reactive group or latent reactive,particularly where the reactivity of the two reactive groups isorthogonal, such as where one is an amine reactive group and the otheris a protected thiol reactive group.

In an embodiment, where the diazo-compound of formula I comprises areactive group and/or a latent reactive group, the invention providesbifunctional or trifunctional and particularly heterobifunctional orheterotrifunctional reagents for bonding the diazo-moiety of thecompound of formula to various R_(M) or R_(M1) groups. In an embodiment,the reactive group or latent reactive group has reactivity that isorthogonal to the diazo-group of the diazo-compound of formula I. Inspecific embodiments, the diazo-compound comprises an amine reactivegroup. In specific embodiments, the diazo-com pound comprises a latentamine reactive group. In specific embodiments, the diazo-compoundcomprises an amine reactive group other than the diazo group and alatent amine reactive group. In specific embodiments, the diazo-compoundcomprises a thiol reactive group. In specific embodiments, thediazo-compound comprises an amine reactive group other than a diazogroup and a thiol reactive group, either of which is a latent reactivegroup. In specific embodiments, the diazo-com pound comprises an R_(M)or R_(M1) group that is a carboxylate reactive group or a latentcarboxylate reactive group (i.e., a protected carboxylate reactivegroup).

The invention also provides compounds of formula II:

where R_(A) is defined as for formula I above and wherein AC representsthe leaving group of an activated ester. Compounds of formula II areuseful at least as reagents in the preparation of the compounds offormula I. Activated esters include among others, N-hydroxysuccinimideesters (NHS esters), N-hydroxysulfosuccinimide esters (sulfo-NHSesters), N-hydroxyphthalimide esters, phenyl esters where the phenylgroup is substituted with one or more electron withdrawing groups,optionally substituted alkyl or aryl sulfonate esters (e.g., tosylesters, mesyl esters, or triflate esters).

Electron withdrawing groups include halogens and nitro groups, forexample. Specific activated esters are fluorinated, chlorinated orbrominated phenyl esters or nitro-substituted phenyl esters. Morespecifically, p-F phenyl; meta, meta-difluorophenyl; meta, meta,para-trifluorophenyl; pentafluorophenyl; p-nitro phenyl; p-chlorophenyl;and p-bromophenyl activated esters can be employed.

In specific embodiments of formula II, R_(A) represents substitution ofthe indicated ring with one to five non-hydrogen substituents on thephenyl ring (any remaining ring positions carrying hydrogens), whereinthe non-hydrogen substituents are selected from the group consisting ofalkyl, cycloalkyl, alkoxy, cycloalkoxy, alkenyl, alkenyloxy,aryl, aryloxy, arylalkyl, arylalkyl oxy, halogen, haloalkyl, haloalkoxy,heterocyclyl, sulfhydryl (—SH), thioalky (—S-alkyl), —NH₂ and—NH—CO—R_(P), where the alkyl, cycloalkyl, alkoxy, cycloalkoxy, alkenyl,alkenyloxy, aryl, aryloxy, arylalkyl, arylalkyloxy and heterocyclylgroups are optionally substituted with 1-5 non-hydrogen substituentsselected from alkyl, alkoxy, halogen, haloalkyl or haloalkoxy groups andR_(P) is hydrogen, an alkyl group or R_(M1), where R_(M1) is as definedfor formula I. In specific embodiments of formula II, R_(A) is in thepara position on the phenyl ring. In specific embodiments of formula II,R_(A) is p-alkyl. In specific embodiments of formula II, R_(A) isp-methyl. In specific embodiments of formula II, R_(A) is p-alkyloxy. Inspecific embodiments of formula II, R_(A) is p-methoxy.

In specific embodiments of formula II, the invention provides reagentsof formula IIA:

where R_(A) is as defined for formula I, and E is hydrogen or —SO₃-(sulfo) salt (e.g., a sodium salt). In specific embodiments of formulaIIA, R_(A) is in the para position on the phenyl ring. In specificembodiments of formula IIA, R_(A) is p-alkyl. In specific embodiments offormula IIA, R_(A) is p-methyl. In specific embodiments of formula IIA,R_(A) is p-alkyloxy. In specific embodiments of formula IIA, R_(A) isp-methoxy. In related specific embodiments of compounds of formula IIAthe NHS ester group can be replaced with an N-hydroxyphthalimide estergroup.

Compounds of formula II and more specifically of formula IIA are atleast useful in the preparation of compounds of formula I. Asillustrated in the examples, the compounds of formula II and IIA can bereacted with amines to generate compounds of formula I.

The invention provides a method for esterifying one or more carboxylicacid groups in an organic or biological molecule which comprisescontacting the organic or biological molecule with a diazo compound offormula I. In a specific embodiment, the reaction is carried out in anaqueous solution. In a specific embodiment, the reaction is carried outin a water/organic solvent mixture. In specific embodiments, the organicsolvent is acetonitrile, methanol, ethanol, t-butanol,dimethylsulfoxide, THF, or related ethers. In specific embodiments, theorganic solvent is acetonitrile. In specific embodiments, the reactionis carried out in solvent containing up to 70% of buffer with organicsolvent. In specific embodiments, the reaction is carried out in solventcontaining from 10-70% (by volume) of water or buffer with organicsolvent. In specific embodiments, the reaction is carried out in solventcontaining from 0.1-10% organic solvent in water or buffer. In specificembodiments, the reaction is carried out in an organic solvent selectedfrom acetonitrile, methanol, ethanol, t-butanol, dimethylsulfoxide, THFor related ethers. The composition of the solvent is dependent upon thesolubility of the diazo-compound in water. In a specific embodiment,dependent upon the solubility of the diazo-compound, the reaction iscarried out in buffered aqueous solution. In a specific embodiment, thereaction is carried out at a pH ranging from 5 to 7 and more preferably5.5 to 6.5. In a specific embodiment, the reaction is carried out at atemperature ranging from about room temperature to about 40° C. In aspecific embodiment, the reaction is carried out at ambient temperature.In a specific embodiment, the reaction is carried out at a temperatureranging from 30-37° C. In a specific embodiment, the reaction is carriedout at a temperature ranging from 25-30° C.

Esterification employing diazo compounds of formula I can, dependentupon R_(M) and/or R_(M1), facilitate labeling, cell targeting, and/orcell penetration of the species (e.g., protein) which is esterified.Compounds of formula I where R_(M) and/or R_(M1) is or comprises areactive group or a latent reactive group can be employed asbifunctional or trifunctional reagents to bond other R_(M) and/or R_(M1)groups which are, for example, labels, cell penetrating groups, or celltargeting groups to the diazo-moiety of formula I. More specifically,compounds of formula I where R_(M) and/or R_(M1) is or comprises areactive group or a latent reactive group can be employed asheterobifunctional or heterotrifunctional reagents where reactive andlatent reactive groups have orthogonal reactivity.

Thus, the invention further provides a method for labeling a molecule(having one or more carboxylate groups, particularly a biologicalmolecule) by covalently bonding a label to the molecule by esterifyingthe carboxylate group(s) of the molecule with a diazo-compound offormula I wherein R_(M) and/or R_(M1) is or comprises a label,particularly a fluorescent label, an isotopic label, a radiolabel, animaging agent, or a quantum dot.

Thus, the invention further provides a method for enhancing cellularuptake of a cargo molecule (having one or more carboxylate groups) bycovalently bonding cell penetrating groups to the cargo molecules byesterifying the cargo molecule with a diazo-compound of formula Iwherein R_(M) is or comprises a cell penetrating group, particularly aguanidinium, positively charged amine, hydrophobic groups such asfluorenyl or pyrene, which are optionally bonded via an -alkylene-CO₂-(e.g., pyrenebutyrate), optionally substituted fluorenyl group oroptionally substituted phenylboronate.

Thus, the invention further provides a method for targeting of a cargomolecule (having one or more carboxylate groups) by covalently bonding acell targeting group to the cargo molecule by esterifying the cargomolecule with a diazo-compound of formula I wherein R_(M) and/or R_(M1)is or comprises a cell targeting group, particularly a ligand for acell-surface receptor (e.g., a steroid, folic acid, substance P, or theRGD tripeptide) or other targeting species such as nuclear localizationpeptides.

In a related aspect, the compound of formula I is employed to esterify atargeting group and one or more cargo molecules are otherwise bondedinto the compound of formula I, for example R_(M) or R_(M1) is orcomprises a cargo molecule or both R_(M) and R_(M1) are or comprise acargo molecule. In this embodiment, the cargo molecule is, for example,a protein, polypeptide or peptide other than the targeting group or is acargo molecule other than a protein, polypeptide or peptide. Forexample, in this embodiment the one or more cargo molecules can be oneor more nucleic acids.

The term cargo molecule is used generally herein to refer to anymolecule that it is desired to target to a cell or to introduce into acell. In specific embodiments, cargo molecules are proteins carrying oneor more carboxylate groups. In specific embodiments, cargo molecules arenucleic acids carrying one or more carboxylate groups.

Dependent upon the R_(M) and R_(M1) groups of the compound of formula I,esterification with the compound of formula I provides for a combinationof labeling, enhancing cell penetration or targeting of the speciesesterified. Thus, the invention provides a method for labeling andadding a cell penetrating group to a selected cargo molecule.Additionally, the invention provides a method for labeling and adding atargeting group to a selected cargo molecule. Additionally, theinvention provides a method for adding a targeting group and a cellpenetration group to a selected cargo molecule. Additionally, theinvention provides a method for adding a label, a targeting group and acell penetration group to a selected cargo molecule. These methods areachieved by esterification of the cargo molecule with a compound offormula I herein where R_(M) and R_(M1) are selected to achieve thedesired introduction of label, targeting group or cell penetrationgroup. Thus, the invention provides methods for labeling combined withenhancement of cell penetration, for labeling combined with celltargeting, for combined cell targeting and enhanced cell penetration, orfor combined labeling, enhanced cell penetration and cell targeting.

When R_(M) or R_(M1) is a polymer, such as polyethylene glycol, theinvention provides a method of functionalizing a cargo molecule, such asa protein, with the polymer by esterification employing a compound offormula I. When R_(M) or R_(M1) is polyethylene glycol, the inventionprovides a method of pegylating a cargo molecule, such as a protein, byesterification employing a compound of formula I.

When R_(M) or R_(M1) is biotin or a derivative thereof, the inventionprovides a method for biotinylation of a cargo molecule, such as aprotein, by esterification employing a compound of formula I. When thebiotin derivative is a labelled biotin, the invention provides a methodfor biotinylation and labelling of the cargo molecule, particularly aprotein, polypeptide or peptide.

The invention also relates in part to methods for enhancing cellularuptake of a cargo molecule by esterifying the cargo molecule with adiazo compound of formula I, wherein R_(M) is a cell penetrating group.A number of such cell penetrating groups are known in the art, whichparticularly include certain peptides. In a specific embodiment,cellular uptake includes at least partial uptake into the cytosol.Cellular uptake may be in vivo or in vitro. The method of the inventionis generally useful for the delivery of any desired molecule carryingone or more carboxylate groups into a cell and specifically includesnucleic acids and analogs thereof; nucleotides and analogs thereof;peptides and proteins; drugs (e.g., anticancer drugs, alkylating agents,antimetabolite, cytotoxic agents; antibiotics, and the like); reportermolecules or labels (e.g., fluorescent labels, isotopic labels, imagingagents, quantum dots, and the like). In a specific embodiment, the cargocomprises a quantum dot carrying amine functionality. The cargo moleculecan include combinations of the species listed above, wherein thespecies are bonded to each other, particularly where the species arecovalently bonded to each other. For example, a cargo molecule maycombine a peptide, such as a CPP or a nuclear localizing signal with anucleic acid, or combine a fluorescent, isotopic or other label with anucleic acid and or peptide. In a specific embodiment, the cargomolecule is or comprises a molecule which affects, regulates ormodulates gene expression in the cell, including a molecule whichinhibits or decreases gene expression or a molecule which initiates orenhances gene expression. In a specific embodiment, the cargo moleculeis a peptide or a protein, for example, an enzyme. In specificembodiments for enhancement of cargo molecule uptake, R_(M) isguanidinium, an optionally substituted fluorenyl group or an optionallysubstituted phenylboronate. Diazo compounds of the invention are mostgenerally compounds of formula I with variables as defined above.

Additional exemplary compounds of formula I are described in more detailbelow. It is noted that compounds of formula II and IIA can be employedto synthesize these additional compounds. In specific embodiments, thecompound of formula I can have formula IA:

where R and R_(A) are defined for formula I, y and z are 0 or 1, -Li-and-L2- are divalent linkers having linker structures as defined herein andE is hydrogen or —SO₃— (sulfo) salt (e.g., a sodium salt). Linkers -Liand L2- can in an embodiment comprise 1-20, 1-12, 1-6 or 1-3 carbonatoms and optionally one or more oxygen atoms. In specific embodimentsof formula IA, —L₁— is present and is —CH₂— and —L₂— is absent. Inspecific embodiments of formula IA, R_(A) is in the para position on thephenyl ring. In specific embodiments of formula IA, R_(A) is p-alkyl. Inspecific embodiments of formula IA, R_(A) is p-methyl. In specificembodiments of formula IA, R_(A) is p-alkyloxy. In specific embodimentsof formula IA, R_(A) is p-methoxy.

In more specific embodiments, compounds of the invention have formulaIB:

where variables are as defined above for formula I. In specificembodiments of formula IB, R is hydrogen or methyl. In specificembodiments of formula IB, R_(M) is alkyl having 1-6 carbon atoms. Inspecific embodiments of formula IB, R_(M) is an alkynyl having 3 or 4carbon atoms.

In an embodiment of formula I, R_(M) or R_(M1) is or comprises theguanidinium group of formula III:

or salts thereof,

-   -   where X_(G) and Y_(G), independently, are optional bonding        moieties (b and d independently are 0 or 1) selected from        —NR_(N)—, —O—, —S—, —S—S—, —CO—NR_(N)—, —CO—O—, —NR_(N)—CO—,        —O—CO—, —CO—, —CO—S—, or —S—CO—; and    -   L_(G) is an optional spacer group (c is 0 or 1) having 1 to 10        carbon atoms and optionally 1-5 oxygen or nitrogen atoms. The        guanidinium group can be protonated and be in the form of a salt        with an appropriate anion. In a specific embodiment of formula        III, b is 1 and X_(G) is O. In a specific embodiment of formula        III, c is 1 and L_(G) is —(CH₂)_(G)—, where G is and integer        ranging from 1-12, 1-6 or 1-3 and more specifically G is 2. In a        specific embodiment of formula III, d is 1 and Y_(G) is NH. In a        specific embodiment of formula III, b is 1 and X_(G) is 0, c is        1 and LG is —(CH₂)_(G)—, where G is 2 or 3 and more specifically        where G is 2. In a specific embodiment of formula III, b is 1, c        is 1 and d is 1, X_(G) is 0, YG is NH and LG is —(CH₂)_(G)—,        where G is 2 or 3 and more specifically where G is 2. In        specific embodiments of compounds of formula I, wherein R_(M1)        is or comprises the guanidinium group of formula III, R is        hydrogen or methyl and R_(M) is an alkyl group having 1-6 carbon        atoms or R_(M) is a alkynyl group having 3-4 carbon atoms. In        specific embodiments of compounds of formula I, wherein R_(M) is        or comprises the guanidinium group of formula III, R is hydrogen        or methyl and R_(A) is an alkyl group having 1-6 carbon atoms,        and more specifically is a methyl group, substituted at the para        position of the phenyl ring.

In a specific embodiment, the reagent of formula I, having R_(M) orR_(M1) that is or comprises the guanidinium of formula III, can beprepared, employing the amine of formula IV:

where G is 1-12, 1-6, or 1-3 and more specifically where G is 2.

In a specific embodiment of formula I, R_(M) or R_(M1) is or comprisesthe fluorenyl group of formula V:

and salts thereof, wherein:

-   -   X_(F) is an optional bonding moiety (g is 0 or 1) selected from        —NR_(N)—, —O—, —S—, —S—S—, —CO—NR_(N)—, —CO—O—, —NR_(N)—CO—,        —O—CO—, —CO—, —CO—S—, or —S—CO—;    -   L_(F) is an optional spacer group (f is 0 or 1) having 1 to 10        carbon atoms and optionally 1-5 oxygen or nitrogen atoms;    -   R_(F) is hydrogen or an alkyl group; and    -   R₃-R₁₀ are selected from hydrogen, alkyl, alkoxy, alkenyl,        alkenoxy, alkynyl, alkynoxy, aryl, aryl oxy, alkylaryl,        alkylaryloxy, arylalkyl, arylalkyloxy, heteroaryl,        heteroaryloxy, carbocyclic, carbocyclyloxy, heterocyclic or        heterocyclyloxy groups each of which is optionally substituted;        or    -   R₃-R₁₀ are selected from non-hydrogen substituents, including        halogens (e.g., Br-, I-, Cl- , F-), hydroxyl (—OH), nitro groups        (—NO₂) , cyano (—CN), isocyano (—NC), thiocyano (—CCN),        isothiocyano (—NCS), sulfuryl (—SO₂), —N(R′)₂, —COR′, —COOR′,        —CON(R′)₂, —NR′—CO—R′, —NR′—CO—N(R′)₂-, —CO—SR′, —SO₂—NR′₂,        —OR′, or —SR′, where each R′, independently, is selected from        hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl,        heterocyclic groups, each of which groups is optionally        substituted particularly with one or more halogen, hydroxyl,        amino, alkylamino, or dialkylamino groups; or two of R₃-R₁₀ are        linked together to form an optionally substituted carbocyclic,        aryl, heterocyclic or heteroaryl ring wherein one or two carbons        of the ring can be replaced with —CO— and the carbocyclic or        heterocyclic rings can be saturated or unsaturated.

In a specific embodiment, all of R₃-R₁₀ are hydrogens. In a specificembodiment, all except one of R₃-R₁₀ are hydrogens. In a specificembodiment, one or more of R₃-R₁₀ are selected from hydrogen, alkylgroups having 1-3 carbon atoms, halogens, —N(R′)₂, —COR′, —COOR′,—CON(R′)₂, —NR′—CO—R′, —NR′—CO—N(R′)₂—, —CO—SR′, —SO₂—NR′₂, —OR′, or—SR′, where each R′, independently, is selected from hydrogen, alkyl,alkenyl, alkynyl, aryl, heteroaryl, heterocyclic groups, each of whichgroups is optionally substituted particularly with one or more halogen,hydroxyl, amino, alkylamino, or dialkylamino groups. In a specificembodiment, one or more of R₃-R₁₀ is a —NR′—CO—R′ group.

In specific embodiments of the R_(M) or R_(M1) group of formula V,R₃-R₁₀ are independently selected from hydrogen, halogen, or alkylgroups having 1-3 carbon atoms. In specific embodiments, R₃-R₁₀ areindependently selected from hydrogen, chlorine, bromine, iodine,fluorine or alkyl groups having 1-3 carbon atoms. In specificembodiments, R₃-R₁₀ are independently selected from hydrogen, halogen,or methyl groups. In specific embodiments, one or two of R₃-R₁₀ areindependently selected from non-hydrogen substituents and the remainingR groups are hydrogens. In specific embodiments, one or two of R₃-R₁₀are selected from halogen, or alkyl groups having 1-3 carbon atoms andthe remaining R groups are hydrogen. In specific embodiments, one or twoof R₃-R₁₀ are selected from halogen, or methyl groups and the remainingR groups are hydrogen.

In a specific embodiment of the R_(M) or R_(M1) group of formula V, oneor both of R₄ and R₉ are —NR′—CO—R′ groups. In specific embodiments, the—NR′—CO—R′ groups are —NH—CO—R′ groups where R′ is an alkyl group or ahaloalkyl group, and more specifically where R′ is a methyl group or atrifluoroethyl group. In specific embodiments, none of R₃—R₁₀ are—NR′—CO—R′ groups. In specific embodiments, none of R₃-R₁₀ are amine oramide groups. In specific embodiments, none of R₃-R₁₀ are isocyanategroups. In specific embodiments, the fluorenyl group can itself exhibitfluorescence.

In specific embodiments of compounds of formula I, wherein R_(M1) is orcomprises the fluorenyl group of formula V, R is hydrogen or methyl andR_(M) is an alkyl group having 1-6 carbon atoms or R_(M) is a alkynylgroup having 3-4 carbon atoms. In specific embodiments of compounds offormula I, wherein R_(M) is or comprises the fluorenyl group of formulaV, R is hydrogen or methyl and R_(A) is an alkyl group having 1-6 carbonatoms, and more specifically is a methyl group, substituted at the paraposition of the phenyl ring.

U.S. published application 2016/0067342 (published Mar. 10, 2016)describes derivatization of cargo molecules with fluorenyl groups forenhancing cellular update of a cargo molecule. This application isincorporated by reference herein in its entirety for descriptions ofmethods of cellular uptake and descriptions of fluorenyl groups for usein the present invention.

In specific embodiments, R_(M) or R_(M1) is a phenylboronic acids suchas those of formulas VIA or VIB:

(noting that VIB is a benzoboroxole structure) or salts thereof,

-   -   where these R_(M) of R_(M1) groups are attached to the compound        of formula I or formula IA through ring positions 3, 4 or 5 or        for formula VIB through ring positions 4 or 5;    -   t is 1 or 2;    -   X_(B) and Y_(B) are optional bonding moieties (u and w are        independently 0 or 1) selected from —NR_(N)—, —O—, —S—, —S—S—,        —CO—NR_(N)—, —CO—O—, —NR_(N)—CO—, —O—CO—, —CO—, —CO—S—, or        —S—CO—;    -   L_(B) is an optional spacer group (v is 0 or 1) having 1 to 10        carbon atoms and optionally 1-5 oxygen or nitrogen atoms;    -   R₁₂-R₁₄, and R₁₆ are independently selected from hydrogen, a        straight-chain or branched aliphatic group having 1-8 carbon        atoms, an alicyclic group, an aryl group, a heterocyclic group,        a heteroaryl group, a —CO₂R₂₀ group, a —O—CO—R₂₀ group, a        —CON(R₂₁)₂ group, a —O—CON(R₂₁)₂ group; a —N(R₂₁)₂ group, a        —OR₂₀ group, a —(CH₂)m—OH group, a —(CH₂)m—N(R₂₁)₂ group, a        halogen, a nitro group, a cyano group, a —SO₂—OR₂₀ group, or two        adjacent R₁₂-R_(14,) and R₁₆, together with the ring carbons to        which they are attached, optionally form a 5-8-member alicyclic,        heterocyclic, aryl or heteroaryl ring moiety, each of which        groups or moieties is optionally substituted;    -   each R₁₇ and R₁₈ is independently selected from hydrogen or a        C1-C3 optionally substituted alkyl group;    -   wherein:    -   each R₂₀ is independently selected from hydrogen, a        straight-chain or branched aliphatic group having 1-8 carbon        atoms, an alicyclic group, an aryl group, a heterocyclic group,        or a heteroaryl group, each of which groups is optionally        substituted;    -   each R₂₁ is independently selected from hydrogen, a        straight-chain or branched aliphatic group having 1-8 carbon        atoms, an alicyclic group, an aryl group, a heterocyclic group,        a heteroaryl group, or where two R21 together with the nitrogen        to which they are attached can form a 5-8 member heterocyclic or        heteroaryl ring moiety, each of which groups or moieties is        optionally substituted;    -   m is an integer from 1-8;    -   wherein optional substitution is substitution by one or more        non-hydrogen substituents selected from halogen; an oxo group        (═O), a nitro group; a cyano group; a C1-C6 alkyl group; a C1-C6        alkoxy group; a C2-C6 alkenyl group; a C2-C6 alkynyl group; a        3-7 member alicyclic ring, wherein one or two ring carbons are        optionally replaced with —CO— and which may contain one or two        double bonds; an aryl group having 6-14 carbon ring atoms; a        phenyl group; a benzyl group; a 5- or 6- member ring        heterocyclic group having 1-3 heteroatoms and wherein one or two        ring carbons are optionally replaced with —CO— and which may        contain one or two double bonds; or a heteroaryl group having        1-3 heteroatoms (N, O or S); a —CO₂R₂₃ group; —OCO—R₂₃ group;        —CON(R₂₄)₂ group; —OCON(R₂₄)₂ group; —N(R₂₄)₂ group; a —SO₂-OR₂₃        group, —OR₂₃ group, —(CH₂)m—OR₂₃ group, —(CH₂)m—N(R₂₄)₂, where m        is 1-8 and each R₂₃ or R₂₄ is independently hydrogen; an        unsubstituted C1-C6 alkyl group; an unsubstituted aryl group        having 6-14 carbon atoms; an unsubstituted phenyl group; an        unsubstituted benzyl group; an unsubstituted 5- or 6- member        ring heterocyclic group, having 1-3 heteroatoms and wherein one        or two ring carbons are optionally replaced with —CO— and which        may contain one or two double bonds; or a heteroaryl group        having 1-3 heteroatoms (N, O or S) and in addition two R24        together with the nitrogen to which they are attached can form a        heterocyclic or heteroaryl ring moiety, each of which groups or        moieties is optionally substituted;    -   each of which R₂₃ and R₂₄ groups is in turn optionally        substituted with one or more unsubstituted C1-C3 alkyl groups,        halogens, oxo groups (═O), nitro groups, cyano groups, —CO₂R₂₅        groups, —OCO—R₂₅ groups, —CON(R₂₆)₂ groups, —OCO—N(R₂₆)₂ groups,        —N(R₂₆)₂ groups, a —SO₂-OR₂₅ group, OR₂₅ groups, —(CH₂)m—OR₂₅        groups, —(CH₂)m—N(R₂₆)₂ where m is 1-8 and each of R₂₅ and R₂₆        independently are hydrogen, an unsubstituted C1-C6 alkyl group;        an unsubstituted aryl group having 6-14 carbon ring atoms; an        unsubstituted phenyl group; an unsubstituted benzyl group, an        unsubstituted 5- or 6- member ring heterocyclic group having 1-3        heteroatoms and wherein a ring carbon is optionally replaced        with —CO— and which may contain one or two double bonds; or a        heteroaryl group having 1-3 heteroatoms (N, O or S) and a total        of 5-14 ring atoms; and in addition two R₂₆ together with the        nitrogen to which they are attached can form an unsubstituted        heterocyclic or heteroaryl ring moiety.

In specific embodiments, —[XB]u—[LB]v—[YB]w— is at ring position 4 informula VIA. In specific embodiments, —[XB]u—[LB]v—[YB]w— is at ringposition 4 in formula VIB.

In specific embodiments of formula I having R_(M1) that is a group offormula VIA or VIB, R is hydrogen or methyl, and R_(M) is an alkylhaving 1-6 carbon atoms or an alkynyl having 3 or 4 carbon atoms. Inspecific embodiments of formula I having R_(M) that is a group offormula VIA or VIB, R is hydrogen or methyl, and R_(A) is an alkylhaving 1-6 carbon atoms substituted at the para position on the phenylring.

In additional embodiments of formula I, R_(M) or R_(M1) is aphenylboronate group of formula VII:

where:

-   -   PB is a phenylboronate group (as defined herein above and as in        U.S. published patent application 2003/0196433 and U.S.        provisional application 62/029,391) 666; Y₄, Y₅ and Y₆ are        independently selected from —O—, —S—, —NRc—, —CO—, —O—CO—,        —CO—O—, —CO—NRc—, —NRc—CO—, —NRc—CO—NRc—, —OCO—NRc—, —NRc—CO—O—,        —N═N—, —N═N—NRc—, —CO—S—, —S—CO—, —S—S—, —SO₂—,        —CRc(OH)—CRc(OH)—, where Rc is hydrogen or C1-C3 alkyl; and    -   L₅ is a divalent spacer moiety, as defined for —L₁— and/or —L₂—        above.

Such phenylboronate groups can be introduced into a compound of formulaI employing a boronation reagent, such as that of formula VIII:

where variables are as defined for formula VII and X₆ is a leaving groupand —Y₆—X₆ together is a reactive group and more specifically is anactivated ester —CO₂AC (as defined in formula II above).

In specific embodiments of compounds of formula I, wherein R_(M1) is orcomprises the group of formula VIII, R is hydrogen or methyl and R_(M)is an alkyl group having 1-6 carbon atoms or R_(M) is an alkynyl grouphaving 3-4 carbon atoms. In specific embodiments of compounds of formulaI, wherein R_(M) is or comprises the group of formula VIII, R ishydrogen or methyl and R_(A) is an alkyl group having 1-6 carbon atoms,and more specifically is a methyl group, substituted at the paraposition of the phenyl ring.

U.S. published patent applications 2003/0196433 and 2016/0024122 areeach incorporated by reference herein in its entirety for description ofstructures of phenylboronate groups useful in this invention forenhancing cell penetration. These references also provide methods formaking phenylboronate groups which can be bonded into compounds offormula 1.

In specific embodiments of formulas herein divalent linkers are selectedfrom the following divalent moieties:

-   -   —Y1—L₁—Y3—, where Y1 and Y3 are optional and may be the same or        different;    -   —Y1—L₁—L2—Y3—, where Y1 and Y3 are optional and may be the same        or different and L₁ and L₂ are different; or        —Y1—L₁—[L₂—Y2]y—L₃—Y3—, where Y1 and Y3 are optional, Y1, Y2 and        Y3 may be the same or different, L₁ and L₃ are optional and L₁,        L₂ and L₃ may be the same or different and y is an integer        indicating the number of repeats of the indicated moiety;    -   wherein each L₁-L₃ is independently selected from an optionally        substituted divalent aliphatic, alicyclic, heterocyclic, aryl,        or heteroaryl moiety having 1 to 30 atoms and each Y1, Y2 and Y3        is independently selected from: —O—, —S—, —NRc—, —CO—, —O—CO—,        —CO—O—, —CO—NRc—, —NRc—CO—, —NRc—CO—NRc—, —OCO—NRc—, —NRc—CO—O—,        —N═N—, —N═N—NRc—, —CO—S—, —S—CO—, —S—S—, —SO₂—,        —CRc(OH)—CRc(OH)—, where Rc is hydrogen or C1-C3 alkyl.

In specific embodiments, divalent linkers are selected from:

-   -   alkylene linkers (—(CH₂)_(y)—) wherein y is 1-12, and preferably        1-4;    -   alkoxyalkyl linkers —[(CH₂)_(q)—O—(CH₂)_(r)]_(a)— wherein q and        r are zero or integers from 1-4, preferably 0, 1, 2 or 3, as        long as one of q and r is not zero, and a is 1-6, preferably        2-4; or    -   aminoalkyl linkers —[(CH₂)_(s)—NR_(N)—(CH₂)_(t)]_(b)— wherein        R_(N) is hydrogen or a C1-C3 alkyl group, s and t are 0 or        integers from 1-4, and are preferably 0, 1 or 2 as long as one        of s and t is not zero, and b is 1-3 and preferably is 1.

In specific embodiments of formulas VII and VIIII, L₅ is —(CH₂)₂—.

In a specific embodiment of the reagent of formula VIII, Y₆—X₆ togetheris a reactive group that reacts with one or more of an amine group, acarboxylic acid group or ester thereof, a sulfhydryl group, a hydroxylgroup, an azide group, a thioester group, a phoshinothioester group, analdehyde group or a ketone group of an amino acid, peptide or protein.

In specific embodiments of formulas VIII, the boronation reagent hasformula IX:

where p-PB is a phenylboronate with the boron in the para position withrespect to the —CH₂—O—, and X₆ is a leaving group. Other boronationagents useful in the present invention are described in U.S. publishedapplication 20160067342, which is incorporated by reference herein fordescriptions of additional phenylboronate groups of formula VII andreagents of formula VIII.

In an embodiment, R_(M) or R_(M1) comprises or is a reactive group andmore specifically an amine-reactive group. In specific embodiments,R_(M) is an amine-reactive group or a spacer moiety substituted with anamine-reactive group for forming one or more amide bonds to a cargomolecule comprising one or more amine group. In specific embodiments,R_(M) comprises or is a latent reactive group or a spacer moietysubstituted with a latent reactive group, which latent reactive groupdoes not react with any reactive group in the compound of formula I, orin any other group in the in compound, and which is selectivelyreactive, or can be selectively activated for reaction, whenappropriate. A latent reactive group can, for example, be activated forreaction inside of a cell for example by enzyme action inside of a cell.A latent reactive group can for example be activated by action of anesterase, for example after the compound of formula I is delivered to acell. In specific embodiments, R_(M) is a spacer moiety substituted witha reactive group. More specifically, R_(M) is a spacer moiety comprisinga latent reactive group and substituted with a reactive group forforming a bond to a cargo molecule wherein the latent reactive groupdoes not react with the reactive group or the cargo molecule and can beselectively reacted or activated for reaction after the cargo moleculeis bonded to the compound of formula I. In specific embodiments, thereactive group of this R_(M) is an amine-reactive group. In specificembodiments, the compound of formula I comprises a reactive group and alatent reactive group.

In a specific embodiment herein, esterification employing a compound offormula I can be employed to covalently bond, via ester formation, acargo molecule to a protein or polypeptide. In this case the cargomolecule is desired to be targeted to a cell, for example, by theprotein, polypeptide or peptide to which it is covalently bound. In thisembodiment, the protein, polypeptide or peptide to which the compound offormula I is esterified functions for cell targeting. The targetingprotein, polypeptide or peptide can be an antibody or functionalfragment thereof. The protein, polypeptide or peptide can be a ligandfor a cell surface receptor. The cargo molecule can itself be a protein,polypeptide or peptide other than the targeting protein, polypeptide orpeptide. The cargo molecule can be a species other than a protein,polypeptide or peptide. The cargo molecule can, employing theesterification methods described herein, further comprise a label and/ora cell penetrating group. In this embodiment, the esterified protein orpolypeptide is contacted with cells for enhanced uptake into cells.

In an embodiment, R_(M) or R_(M1) is or comprises a polymer which canfunction for protection of a protein in the bloodstream or to enhancepharmokinetics of the protein. In an embodiment, R_(M) or R_(M1) is orcomprises polyethylene glycol. In more specific embodiments, thepolyethylene glycol has average molecular weight ranging from 200 to10,000. In more specific embodiments, the polyethylene glycol hasaverage molecular weight ranging from 1,000 to 10,000. In more specificembodiments, the polyethylene glycol has average molecular weightranging from 2,000 to 10,000. In more specific embodiments, thepolyethylene glycol has average molecular weight ranging from 2,000 to6,000. In more specific embodiments, the polyethylene glycol has numberaverage molecular weight (Mn) ranging from 200 to 10,000, 1,000 to10,000, 2,000 to 10,000, or from 2,000 to 6,000. Functionalizedpolyethylene glycol polymers useful for preparation of compounds offormula 1 are commercially available or can be prepared by well-knownmethods. See for example, The Sigma-Aldrich Catalogue.

In an embodiment, R_(M) or R_(M1) is or comprises biotin or a derivativethereof. In specific embodiments, the biotin derivative is any biotinderivative known in art and useful for biotinylation of a chemical orbiochemical species, such as a protein, polypeptide or peptide. Biotinderivatives include labelled biotin, such as radiolabelled biotin,isotopically labelled biotin, biotin labelled with a fluorescent orother dye, or the like. Functionalized biotins, such as aminefunctionalized biotin, useful in the preparation of compounds of formulaI containing biotin or a derivative thereof are known in the art and/orcommercially available. (See, for example, Sigma-Aldrich Catalogue).Esterification of a cargo molecule, such as a protein with a compound offormula I which comprises biotin or a derivative thereof (e.g., alabelled biotin) can provide for biotinylation of the cargo molecule forany know purpose. For example, functionalization of a protein withbiotin can be employed for protein capture or isolation, for example,for protein pull-down or for biotin affinity purification. Thus, theinvention provides a method for biotinylating a cargo molecule or moresimply a protein, polypeptide or peptide employing a compound of formulaI where R_(M) or R_(M1) is or comprises biotin or a derivative thereof.

Diazo compounds of formula I can be synthesized in view of the examplesprovided herein and in U.S. Pat. No. 8,350,014 and in further view ofwhat is well-known in the art. Methods herein can be routinely adaptedby choice of starting materials, solvents and reagents as known in theart to prepare compounds of formula I not specifically exemplified.R_(M) and R_(M1) groups comprising labels, cell penetrating groups, orcell targeting groups can for example be in introduced into thecompounds of formula I employing bioconjugation methods as found inHermanson, G. T. Bioconjugation Techniques (2^(nd) Ed.) 2008 AcademicPress/Elsevier London, UK. This reference also contains detaileddescriptions of homobifunctional and heterobifunctional crossing linkingreagents which can be employed to covalently attach a R_(M1) and R_(M)groups in compounds of formula I. U.S. Pat. No. 8,350,014 isincorporated by reference herein in its entirety for descriptions ofsynthesis of diazo compounds.

Diazo compounds of formula I carrying one or more reactive or latentreactive groups can be prepared in view of methods herein and what iswell-known in the art. For example, methods as described in Josa-Cullere(2014) [39] and Ma, M. et al. (2005) [40] can be employed to preparecompounds of formula I having NHS esters.

The terms alkyl or alkyl group refer to a monoradical of astraight-chain or branched saturated hydrocarbon. Alkyl groups includestraight-chain and branched alkyl groups. Unless otherwise indicatedalkyl groups have 1-20 carbon atoms (C1-C20 alkyl groups) and preferredare those that contain 1-10 carbon atoms (C1-C10 alkyl groups) and morepreferred are those that contain 1-6 carbon atoms (C1-C6 alkyl groups)and those that contain 1-3 carbon atoms (C1-C3 alkyl groups) Alkylgroups are optionally substituted with one or more non-hydrogensubstituents as described herein. Exemplary alkyl groups include methyl,ethyl, n-propyl, iso-propyl, n-butyl, s-butyl, t-butyl, n-pentyl,branched-pentyl, n-hexyl, branched hexyl, all of which are optionallysubstituted. Substituted alkyl groups include fully halogenated orsemihalogenated alkyl groups, such as alkyl groups having one or morehydrogens replaced with one or more fluorine atoms, chlorine atoms,bromine atoms and/or iodine atoms. Substituted alkyl groups includefully fluorinated or semifluorinated alkyl.

A carbocyclyl group is a group having one or more saturated orunsaturated carbon rings. Carbocyclyl groups, for example, contain oneor two double bonds. One or more carbons in a carbocyclic ring can be—CO— groups. Carbocyclyl groups include those having 3-12 carbon atoms,and optionally replacing 1 or 2 carbon atoms with a —CO— group andoptionally having 1, 2 or 3 double bonds. Carbocyclyl groups includethose having 5-6 ring carbons. Carbocyclyl groups can contain one ormore rings each of which is saturated or unsaturated. Carbocyclyl groupsinclude bicyclic and tricyclic groups. Preferred carbocyclic groups havea single 5- or 6-member ring. Carbocyclyl groups are optionallysubstituted as described herein. Specifically, carbocyclic groups can besubstituted with one or more alkyl groups. Carbocyclyl groups includeamong others cycloalkyl and cycloalkenyl groups.

Cycloalkyl groups include those which have 1 ring or which are bicyclicor tricyclic. In specific embodiments, cycloalkyl groups have 1 ringhaving 5-8 carbon atoms and preferably have 5 or 6 carbon atoms.

Cycloalkenyl groups include those which have 1 ring or which arebicyclic or tricyclic and which contain 1-3 double bond. In specificembodiments, cycloalkenyl groups have 1 ring having 5-8 carbon atoms andpreferably have 5 or 6 carbon atoms and have one double bond.

Alkenyl groups include monovalent straight-chain, branched and cyclicalkenyl groups which contain one or more carbon-carbon double bonds.Unless otherwise indicated alkenyl groups include those having from 2 to20 carbon atoms. Alkenyl groups include those having 2 to 4 carbon atomsand those having from 5-8 carbon atoms. Cyclic alkenyl groups includethose having one or more rings wherein at least one ring contains adouble bond. Cyclic alkenyl groups include those which have 1, 2 or 3rings wherein at least one ring contains a double bond. Cyclic alkenylgroups also include those having 3-10 carbon atoms. Cyclic alkenylgroups include those having a 5-, 6-, 7-, 8-, 9- or 10-member carbonring and particularly those having a 5- or 6-member ring. The carbonrings in cyclic alkenyl groups can also carry straight-chain or branchedalkyl or alkenyl group substituents. Cyclic alkenyl groups can includebicyclic and tricyclic alkyl groups wherein at least one ring contains adouble bond. Alkenyl groups are optionally substituted with one or morenon-hydrogen substituents as described herein. Specific alkenyl groupsinclude ethylene, propenyl, cyclopropenyl, butenyl, cyclobutenyl,pentenyl, pentadienyl, cyclopentenyl, cyclopentadienyl, hexylenyl,hexadienyl, cyclohexenyl, cyclohexadienyl, including all isomers thereofand all of which are optionally substituted. Substituted alkenyl groupsinclude fully halogenated or semihalogenated alkenyl groups.

Alkynyl groups include mono-valent straight-chain, branched and cyclicalkynyl group which contain one or more carbon-carbon triple bonds.Unless otherwise indicated alkynyl groups include those having from 2 to20 carbon atoms. Alkynyl groups include those having 2 to 4 carbon atomsand those having from 5-8 carbon atoms. Cyclic alkynyl groups includethose having one or more rings wherein at least one ring contains atriple bond. Cyclic alkynyl groups include those which have 1, 2 or 3rings wherein at least one ring contains a triple bond. Cyclic alkynylgroups also include those having 3-10 carbon atoms. Cyclic alkynylgroups include those having a 5-, 6-, 7-, 8-, 9- or 10-member carbonring and particularly those having a 5- or 6-member ring.

The carbon rings in cyclic alkynyl groups can also carry straight-chainor branched alkyl, alkenyl or alkynyl group substituents. Cyclic alkynylgroups can include bicyclic and tricyclic alkyl groups wherein at leastone ring contains a triple bond. Alkynyl groups are optionallysubstituted with one or more non-hydrogen substituents as describedherein.

An alkoxy group is an alkyl group (including cycloalkyl), as broadlydiscussed above, linked to oxygen, a monovalent —O-alkyl group. Anaryloxy group is an aryl group, as discussed above, linked to an oxygen,a monovalent —O-aryl. A heteroaryloxy group is a heteroaryl group asdiscussed above linked to an oxygen, a monovalent —O-heteroaryl.Alkenoxy, alkynoxy, alicycloxy, heterocycloxy groups are analogouslydefined. All of such groups are optionally substituted.

An aliphatic group as used herein refers to a monovalent non-aromatichydrocarbon group which include straight chain, branched, or cyclichydrocarbon groups which can be saturated or unsaturated with one ormore double bonds or one or more triple bonds. Aliphatic groups maycontain portions which are straight-chain or branched in combinationwith one or more carbon rings. Carbon rings of aliphatic groups maycontain one or more double bonds or one or more triple bonds. Carbonrings of aliphatic groups can contain 3- to 10-membered rings. Suchcarbon rings may be fused and may be bicyclic or tricyclic. Aliphaticgroups are optionally substituted with one or more non-hydrogensubstituents where optional substituents are described herein. Unlessotherwise specified, an aliphatic group can contain 1-20 carbon atoms orcan contain 1-10 carbon atoms. Aliphatic groups include those containing1-3, 1-6, and 1-8 carbon atoms. Aliphatic groups include, among others,alicyclic groups, alkyl groups, alkenyl groups and alkynyl groups.

Heteroaliphatic groups refer generally to aliphatic groups having 1 ormore heteroatoms (other than C and H). Specifically heteroatoms ofheteroaliphatic groups are selected from N, P, B, O or S. In morespecific embodiments, heteroaliphatic groups contain one or moreoxygens, nitrogen or sulfur atoms.

An alicylic group as used herein refers to a monovalent non-aromaticcyclic hydrocarbon group which can be saturated or unsaturated with oneor more double bonds or one or more triple bonds. Alicyclic ringsinclude those containing 3- to 10-membered carbon rings. Alicyclicgroups include those containing one, two, three or more rings which maybe fused or linked by straight chain or branched alkylene, alkenylene oralkynylene moieties. Alicyclic groups include bicyclic and tricyclicrings. Alicyclic groups include those in which one or more carbon ringsare substituted with a straight-chain or branched alkyl, alkenyl oralkynyl group. To satisfy valence requirements, a ring atom may besubstituted with hydrogen or optionally with non-hydrogen substituentsas described herein. One or more carbons in an alicyclic group can be—CO— groups, i.e. a carbon can be substituted with an oxo (═O) moiety.Alicyclic groups are optionally substituted with one or morenon-hydrogen substituents where optional substituents are describedherein. Unless otherwise specified, an alicyclic group can contain 3-20carbon atoms or can contain 3-12 carbon atoms. Alicyclic groups includethose containing 3-6 and 3-8 carbon atoms. Alicyclic groups includeamong others cycloalkyl, cycloalkenyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl andcyclohexadienyl groups, all of which are optionally substituted.

The number of carbon atoms in a given group, such as an alkyl group, canbe indicated herein using the expression “Cm” where m is the number ofcarbon atoms. Thus, the expression “Cm1-Cm2” modifying a given chemicalgroup indicates that the group can contain from m1 to m2 carbon atoms.For example, a C1-C6 alkyl group contains 1 to 6 carbon atoms, exclusiveof carbons in any substituent on the alkyl group. Similar expressionscan be used to indicate the number of atoms of N (nitrogen), O (oxygen)or other elements in a given group.

A heterocyclyl (or heterocyclic) group is a group having one or moresaturated or unsaturated carbon rings and which contains one to threeheteroatoms (e.g., N, O or S) per ring. These groups optionally containone, two or three double bonds. To satisfy valence requirement, a ringatom may be substituted as described herein. One or more carbons in theheterocyclic ring can be —CO-groups. Heterocyclyl groups include thosehaving 3-12 carbon atoms, and 1-6, heteroatoms, wherein 1 or 2 carbonatoms are replaced with a —CO-group. Heterocyclyl groups include thosehaving 3-12 or 3-10 ring atoms of which up to three can be heteroatomsother than carbon. Heterocyclyl groups can contain one or more ringseach of which is saturated or unsaturated. Heterocyclyl groups includebicyclic and tricyclic groups. Preferred heterocyclyl groups have 5- or6-member rings. Heterocyclyl groups are optionally substituted asdescribed herein. Specifically, heterocyclic groups can be substitutedwith one or more alkyl groups. Heterocyclyl groups include those having5- and 6-member rings with one or two nitrogens and one or two doublebonds. Heterocyclyl groups include those having 5- and 6-member ringswith an oxygen or a sulfur and one or two double bonds. Heterocyclylgroup include those having 5- or 6-member rings and two differentheteroatom, e.g., N and O, O and S or N and S. Specific heterocyclylgroups include among others among others, pyrrolidinyl, piperidyl,piperazinyl, pyrrolyl, pyrrolinyl, furyl, thienyl, morpholinyl,oxazolyl, oxazolinyl, oxazolidinyl, indolyl, triazoly, and triazinylgroups.

Aryl groups include groups having one or more 5- or 6-member aromaticrings. Aryl groups can contain one, two or three, 6-member aromaticrings. Aryl groups can contain two or more fused aromatic rings. Arylgroups can contain two or three fused aromatic rings. Aryl groups areoptionally substituted with one or more non-hydrogen substituents.Substituted aryl groups include among others those which are substitutedwith alkyl or alkenyl groups, which groups in turn can be optionallysubstituted. Specific aryl groups include phenyl groups, biphenylgroups, and naphthyl groups, all of which are optionally substituted asdescribed herein. Substituted aryl groups include fully halogenated orsemihalogenated aryl groups, such as aryl groups having one or morehydrogens replaced with one or more fluorine atoms, chlorine atoms,bromine atoms and/or iodine atoms. Substituted aryl groups include fullyfluorinated or semifluorinated aryl groups, such as aryl groups havingone or more hydrogen replaced with one or more fluorine atoms.

Heteroaryl groups include groups having one or more aromatic rings inwhich at least one ring contains a heteroatom (a non-carbon ring atom).Heteroaryl groups include those having one or two heteroaromatic ringscarrying 1, 2 or 3 heteroatoms and optionally have one 6-member aromaticring. Heteroaryl groups can contain 5-20, 5-12 or 5-10 ring atoms.Heteroaryl groups include those having one aromatic ring contains aheteroatom and one aromatic ring containing carbon ring atoms.Heteroaryl groups include those having one or more 5- or 6-memberaromatic heteroaromatic rings and one or more 6-member carbon aromaticrings. Heteroaromatic rings can include one or more N, O, or S atoms inthe ring. Heteroaromatic rings can include those with one, two or threeN, those with one or two O, and those with one or two S, or combinationsof one or two or three N, O or S. Specific heteroaryl groups includefuryl, pyridinyl, pyrazinyl, pyrimidinyl, quinolinyl, and purinylgroups. In specific embodiments herein aryl groups contain noheteroatoms in the aryl rings. Aryl including heteroaryl groups areoptionally substituted.

Heteroatoms include O, N, S, P or B. More specifically heteroatoms areN, O or S. In specific embodiments, one or more heteroatoms aresubstituted for carbons in aromatic or carbocyclic rings. To satisfyvalence any heteroatoms in such aromatic or carbocyclic rings may bebonded to H or a substituent group, e.g., an alkyl group or othersubstituent.

Heteroarylalkyl groups are alkyl groups substituted with one or moreheteroaryl groups wherein the alkyl groups optionally carry additionalsubstituents and the aryl groups are optionally substituted.

Alkylaryl groups are aryl groups substituted with one or more alkylgroups wherein the alkyl groups optionally carry additional substituentsand the aryl groups are optionally substituted. Specific alkylarylgroups are alkyl-substituted phenyl groups such as methylphenyl.

Arylalkyl groups are alkyl groups substituted with one or more arylgroups, typically one aryl group. The aryl group is optionallysubstituted. Specific arylakly groups include benzyl, optionallysubstituted benzyl, phenethyl, and optionally substituted phenethyl.

Alkylheteroaryl groups are heteroaryl groups substituted with one ormore alkyl groups wherein the alkyl groups optionally carry additionalsubstituents and the aryl groups are optionally substituted.

An alkoxy group is an alkyl group, as broadly discussed above, linked tooxygen (R_(alkyl)-O—). An aryloxy group is an aryl group, as discussedabove, linked to an oxygen (R_(aryl)-O—). A heteroaryloxy group is aheteroaryl group as discussed above linked to an oxygen(R_(heteroaryl)-O—). A carbocyclyloxy group is an carbocyclyl group, asbroadly discussed above, linked to oxygen (R_(carbocyclyl)-O—). Aheterocyclyloxy group is an carbocyclyl group, as broadly discussedabove, linked to oxygen (R_(heterocyclyl)-O—).

An acyl group is an R′—CO group where R′ in general is a hydrogen, analkyl, alkenyl or alkynyl, aryl or heteroaryl group as described above.In specific embodiments, acyl groups have 1-20, 1-12 or 1-6 carbon atomsand optionally 1-3 heteroatom, optionally one double bond or one triplebond. In specific embodiments, R is a C1-C6 alkyl, alkenyl or alkynylgroup. cyclic configuration or a combination thereof, attached to theparent structure through a carbonyl functionality. Examples includeacetyl, benzoyl, propionyl, isobutyryl, or oxalyl. The R′ group of acylgroups are optionally substituted as described herein. When R′ ishydrogen, the group is a formyl group. An acetyl group is a CH₃—CO—group. Another exemplary acyl group is a benzyloxy group.

An alkylthio group is an alkyl group, as broadly discussed above, linkedto a sulfur (R_(alkyl)-S—) An arylthio group is an aryl group, asdiscussed above, linked to a sulfur (R_(aryl)-S—). A heteroarylthiogroup is a heteroaryl group as discussed above linked to an sulfur(R_(heteroaryl)-S—). A carbocyclylthio group is an carbocyclyl group, asbroadly discussed above, linked to oxygen (R_(carbocyclyl)-S—). Aheterocyclylthio group is an carbocyclyl group, as broadly discussedabove, linked to oxygen (R_(heterocyclyl)-S—).

The term amino group refers to the species —N(H)₂—. The term alkylaminorefers to the species —NHR″ where R″ is an alkyl group, particularly analkyl group having 1-3 carbon atoms. The term dialkylamino refers to thespecies —NR″₂ where each R″ is independently an alkyl group,particularly an alkyl group having 1-3 carbon atoms.

Groups herein are optionally substituted most generally with one or morealky, alkenyl, alkynyl, and aryl, heteroaryl, carbocyclyl, andheterocyclyl groups can be substituted, for example, with one or moreoxo group, thioxo group, halogen, nitro, cyano, cyanate, azido,thiocyano, isocyano, isothiocyano, sulfhydryl, hydroxyl, alkyl, alkoxy,alkenyl, alkenyloxy, alkynyl, alkynyloxy, aryl, aryloxy, heteroaryl,heteroaryloxy, carbocyclyl, carbocyclyloxy, heterocyclyl,heterocyclyloxy, alkylthio, alkenylthio, alkynylthio, arylthio,thioheteroaryl, thioheteroaryl, thiocarbocyclyl, thioheterocyclyl,—CORs, —COH, —OCORs, —OCOH, —CO—ORs, —CO—OH, —CO—O—CO—Rs, —CON(Rs)₂,—CONHRs, —CONH₂, —NRs—CORs, —NHCORs, —NHRs, —N(Rs)₂, —O—SO₂—Rs, —SO₂—Rs,—SO₂-NHRs, —SO₂—N(Rs)₂, —NRs—SO₂—Rs, —NH—SO₂—Rs, —NRsCO—N(Rs)₂,—NH—CO—NHRs, —O—PO(ORs)₂, —O—PO(ORs)(N(Rs)₂), —O—PO(N(Rs)₂)₂,—N—PO(ORs)₂, —N—PO(ORs)(N(Rs)₂), —P(Rs)₂, —B(OH)₂, —B(OH)(ORs), —B(ORs)₂, where each Rs independently is an organic group and morespecifically is an alkyl, alkenyl, alkynyl, aryl, heteroaryl,carbocyclyl, or heterocyclyl group or two Rs within the same substituentcan together form a carbocyclic or heterocyclic ring having 3 to 10 ringatoms. Organic groups of non-hydrogen substituents are in turnoptionally substituted with one or more halogens, nitro, cyano,isocyano, isothiocyano, hydroxyl, sulfhydryl, haloalkyl, hydroxyalkyl,amino, alkylamino, dialkylamino, arylalkyl, unsubstituted alkyl,unsubstituted alkenyl, unsubstituted alkynyl alkylalkenyl, alkylalkynyl,haloaryl, hydroxylaryl, alkylaryl, unsubstituted aryl, unsubstitutedcarbocylic, halo-substituted carbocyclic, hydroxyl-substitutedcarbocyclic, alkyl-substituted carbocyclic, unsubstituted heterocyclic,unsubstituted heteroaryl, alkyl-substituted heteroaryl, oralkyl-substituted heterocyclic. In specific embodiments, Rs groups ofsubstituents are independently selected from alkyl groups, haloalkylgroups, phenyl groups, benzyl groups and halo-substituted phenyl andbenzyl groups. In specific embodiments, non-hydrogen substituents have1-20 carbon atoms, 1-10 carbon atoms, 1-7 carbon atoms, 1-5 carbon atomsor 1-3 carbon atoms. In specific embodiments, non-hydrogen substituentshave 1-10 heteroatoms, 1-6 heteroatoms, 1-4 heteroatoms, or 1, 2, or 3heteroatoms. Heteroatoms include O, N, S, P, B and Se and preferably areO, N or S.

In specific embodiments, optional substitution is substitution with 1-12(or 1-3 or 1 to 3 or 1 to 6) non-hydrogen substituents. In specificembodiments, optional substitution is substitution with 1-6 non-hydrogensubstituents. In specific embodiments, optional substitution issubstitution with 1-3 non-hydrogen substituents. In specificembodiments, optional substituents contain 6 or fewer carbon atoms. Inspecific embodiments, optional substitution is substitution by one ormore halogen, hydroxyl group, cyano group, oxo group, thioxo group,unsubstituted C1-C6 alkyl group or unsubstituted aryl group. The termoxo group and thioxo group refer to substitution of a carbon atom with a═O or a ═S to form respectively —CO— (carbonyl) or —CS— (thiocarbonyl)groups.

In specific embodiments, non-hydrogen substituents for optionalsubstitution include alkyl, alkoxy, halogen (F, Cl, Br or I andpreferably Cl or F), haloalkyl, or haloalkoxy. In specific embodiments,non-hydrogen substituents for optional substitution include methyl,ethyl, methoxy, ethoxy, F, Cl, and trifluormethyl.

Specific substituted alkyl groups include haloalkyl groups, particularlytrihalomethyl groups and specifically trifluoromethyl groups. Specificsubstituted aryl groups include mono-, di-, tri, tetra- andpentahalo-substituted phenyl groups; mono-, di, tri-, tetra-, penta-,hexa-, and hepta-halo-substituted naphthalene groups; 3- or4-halo-substituted phenyl groups, 3- or 4-alkyl-substituted phenylgroups, 3- or 4-alkoxy-substituted phenyl groups, 3- or4-RsCO-substituted phenyl, 5- or 6-halo-substituted naphthalene groups.More specifically, substituted aryl groups include acetylphenyl groups,particularly 4-acetylphenyl groups; fluorophenyl groups, particularly3-fluorophenyl and 4-fluorophenyl groups; chlorophenyl groups,particularly 3-chlorophenyl and 4-chlorophenyl groups; methylphenylgroups, particularly 4-methylphenyl groups, and methoxyphenyl groups,particularly 4-methoxyphenyl groups.

As to any of the above groups which contain one or more substituents, itis understood, that such groups do not contain any substitution orsubstitution patterns which are sterically impractical and/orsynthetically non-feasible. In addition, the compounds of this inventioninclude all stereochemical isomers arising from the substitution ofthese compounds.

Compounds of the invention may contain chemical groups (acidic or basicgroups) that can be in the form of salts. Exemplary acid addition saltsinclude acetates (such as those formed with acetic acid or trihaloaceticacid, for example, trifluoroacetic acid), adipates, alginates,ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates,borates, butyrates, citrates, camphorates, camphorsulfonates,cyclopentanepropionates, digluconates, dodecylsulfates,ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates,hemisulfates, heptanoates, hexanoates, hydrochlorides (formed withhydrochloric acid), hydrobromides (formed with hydrogen bromide),hydroiodides, 2-hydroxyethanesulfonates, lactates, maleates (formed withmaleic acid), methanesulfonates (formed with methanesulfonic acid),2-naphthalenesulfonates, nicotinates, nitrates, oxalates, pectinates,persulfates, 3-phenylpropionates, phosphates, picrates, pivalates,propionates, salicylates, succinates, sulfates (such as those formedwith sulfuric acid), sulfonates (such as those mentioned herein),tartrates, thiocyanates, toluenesulfonates such as tosylates,undecanoates, and the like.

Exemplary basic salts include ammonium salts, alkali metal salts such assodium, lithium, and potassium salts, alkaline earth metal salts such ascalcium and magnesium salts, salts with organic bases (for example,organic amines) such as benzathines, dicyclohexylamines, hydrabamines[formed with N,N-bis(dehydro-abietyl)ethylenediamine],N-methyl-D-glucamines, N-methyl-D-glucamides, t-butyl amines, and saltswith amino acids such as arginine, lysine and the like. Basicnitrogen-containing groups may be quaternized with agents such as loweralkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides,bromides and iodides), dialkyl sulfates (e.g., dimethyl, diethyl,dibutyl, and diamyl sulfates), long chain halides (e.g., decyl, lauryl,myristyl and stearyl chlorides, bromides and iodides), aralkyl halides(e.g., benzyl and phenethyl bromides), and others.

Salts of the invention include “pharmaceutically acceptable salts” whichrefers to those salts which retain the biological effectiveness andproperties of the free bases or free acids, and which are notbiologically or otherwise undesirable. Pharmaceutically acceptable saltscomprise pharmaceutically-acceptable anions and/or cations.

Compounds of the present invention, and salts thereof, may exist intheir tautomeric form, in which hydrogen atoms are transposed to otherparts of the molecules and the chemical bonds between the atoms of themolecules are consequently rearranged. It should be understood that alltautomeric forms, insofar as they may exist, are included within theinvention.

Additionally, inventive compounds may have trans and cis isomers and maycontain one or more chiral centers, therefore exist in enantiomeric anddiastereomeric forms. The invention includes all such isomers, as wellas mixtures of cis and trans isomers, mixtures of diastereomers andracemic mixtures of enantiomers (optical isomers). When no specificmention is made of the configuration (cis, trans or R or S) of acompound (or of an asymmetric carbon), then any one of the isomers or amixture of more than one isomer is intended. The processes forpreparation can use racemates, enantiomers, or diastereomers as startingmaterials. When enantiomeric or diastereomeric products are prepared,they can be separated by conventional methods, for example, bychromatographic or fractional crystallization. The inventive compoundsmay be in the free or hydrate form. With respect to the variouscompounds of the invention, the atoms therein may have various isotopicforms, e.g., isotopes of hydrogen include deuterium and tritium. Allisotopic variants of compounds of the invention are included within theinvention and particularly included at deuterium and ¹³C isotopicvariants. It will be appreciated that such isotopic variants may beuseful for carrying out various chemical and biological analyses,investigations of reaction mechanisms and the like. Methods for makingisotopic variants are known in the art.

In embodiments of the methods herein a cargo molecule is esterified witha compound of formula I herein and a cell or tissue is contacted withthe esterified cargo molecule. Contacting with a cell or tissue istypically carried out in an aqueous buffer suitable for the cell ortissue. Contacting is typically carried out in an aqueous buffer ofappropriate pH which can be readily selected by one of ordinary skill inthe art. Typically, contacting is carried out at pH ranging from 5 to 8.Contacting can include administration to an organism or individual. Anysuitable form of administration can be employed in the methods herein.The esterified cargo molecules of this invention can, for example, beadministered orally, topically, intravenously, intraperitoneally,subcutaneously, or intramuscularly, in any suitable dosage forms wellknown to those of ordinary skill in the pharmaceutical arts. Theesterified cargo molecules are optionally administered with apharmaceutical carrier selected upon the basis of the chosen route ofadministration and standard pharmaceutical practice, such as, forexample, as described in Remington's Pharmaceutical Sciences, 17thedition, ed. Alfonoso R. Gennaro, Mack Publishing Company, Easton, Pa.(1985), which is incorporated herein by reference in its entirety forsuitable administration and carriers.

Cargo molecules include nucleic acids, peptides, proteins, smallmolecule drugs, reporters and labeling (fluorescent labels or isotopiclabels for example), imaging agents, contrast agents, particles carryingreactive functional groups, quantum dots carrying reactive functionalgroups, among others. In general any cargo molecule that it is desiredto introduce into a cell can be employed in the methods of thisinvention. Cargo molecules include those having a biological activity.In specific embodiments, biological activity of interest of the cargomolecule is retained on esterification or is recovered on selectiveremoval of esterification after delivery to a cell. In a specificembodiment, the esterified cargo molecule retains at least 10% of aselected biological activity of the cargo molecule prior toesterification. In other specific embodiments, the esterified cargomolecule retains at least 50% of a selected biological activity of thecargo molecule prior to esterification. In a further specificembodiment, the esterified cargo molecule retains at least 80% of theactivity of the cargo molecule prior to esterification.

In a specific embodiment, the cargo protein is an enzyme. In a specificembodiment, the cargo protein is glycosylated (i.e., is a glycoprotein).In a specific embodiment, the cargo protein is not glycosylated (i.e.,is not a glycoprotein). In a specific embodiment, the esterified cargopeptide or protein retains at least 10% of a selected biologicalactivity of the protein prior to esterification. In other specificembodiments, esterified cargo peptide or protein retains at least 50% ofa selected biological activity of the protein prior to esterification.In a further specific embodiment, the esterified cargo peptide orprotein retains at least 80% of the activity of the peptide or proteinprior to esterification. Peptides and proteins include those havingenzyme activity.

Cargo peptides include peptide ligands, cytotoxic peptides, bioactivepeptides, diagnostic agents, among others. Cargo peptides include thosehaving 2-1000 amino acids, 2-500 amino acids, 2-250 amino acids, 2-100amino acids, 2-50 amino acids, and 2-25 amino acids and 2-10 aminoacids.

Peptides and proteins include antibodies and functional fragmentsthereof, where the term antibody is used broadly herein. Morespecifically, antibodies include among others, monoclonal antibodiesincluding humanized antibodies, human antibodies, interspeciesantibodies, chimeric antibodies, human monoclonals, humanizedmonoclonals, interspecies antibodies made by any art-known methods.Functional fragments of antibodies include F(ab′)₂, F(ab)₂, Fab′, Fab,Fv, among others, as well as hybrid fragments. Additionally, antibodiesinclude subfragments retaining the hypervariable, antigen-binding regionof an immunoglobulin and preferably having a size similar to or smallerthan a Fab′ fragment. Such fragments and subfragments, including singlechain fragments or multiple chain fragments, which incorporate anantigen-binding site and exhibit antibody function, are known in the artand can be prepared by methods that are well-known in the art, includingby methods of preparing recombinant proteins. Antibodies and fragmentsthereof include therapeutic antibodies which are known in the art [35].This reference is incorporated by reference herein in its entirety fordescriptions of therapeutic antibodies which can be employed in thepresent invention.

In a specific embodiment, the cargo molecule is a nucleic acid which maybe RNA or DNA, or an analog of a nucleic acid which may be a peptidenucleic acid, a locked nucleic acid, or a phosphoramidate-morpholinooligomer. Other art-known nucleic acid analogs include carbamate-linkedDNA, phosphorothioate-linked DNA, 2′-O-methyl RNA,phosphotriester-linked DNA or methylphosphonate-linked DNA. The cargonucleic acid can be single- or double-stranded. The nucleic acid can bean oligonucleotide or analog thereof having 2-100, 2-50 or 2-25 bases.The nucleic acid can be siRNA, microRNA, antisense oligonucleotides,decoy DNA, plasm ids or other nucleic acid structures such asminicircles. Nucleic acids and analogs thereof are available fromcommercial sources, can be isolated from natural source or can beprepared by methods that are well-known in the art.

In a specific embodiment, the esterified cargo nucleic acid retains atleast 10% of a selected biological activity of the nucleic acid prior toesterification. In other specific embodiments, the esterified cargonucleic acid retains at least 50% of a selected biological activity ofthe nucleic acid prior to esterification. In a further specificembodiment, the esterified cargo nucleic acid retains at least 80% ofthe activity of the nucleic acid prior to esterification. In a specificembodiment, the biological activity of the nucleic acid that is retainedis binding to a complementary nucleic acid or binding to anotherbiological molecule (e.g., a peptide or protein).

Cargo nucleic acids include those having 2-1000 bases, 2-500 bases,2-250 bases, 2-100 bases, 2-50 bases, and 2-25 bases and 2-10 bases.Nucleic acids include nucleosides and analogs thereof.

In specific embodiments, cargo molecules include transcription factors(proteins) which affect transcription of DNA to messenger RNA and thusaffect expression of one or more genes. In specific embodiments,transcription factors include one or more DNA-binding domains.Transcription factors include, among others, tumor suppressors. Aspecific transcription factor of potential clinical interest is FOXO3which functions as a trigger for apoptosis (36). One or morediazo-compounds of formula I can be employed to esterify transcriptionfactors, including FOXO transcription factors, and more specificallyFOXO3 to facilitate cell uptake thereof. Employing the reversible diazoesterification reagents herein, esterified groups are removed after celluptake.

In specific embodiments, cargo molecules include proteins that functionas tumor suppessors. For example, cargo molecules include PTEN which isa phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase (Hopkins, etal. 2013, 7) PTEN contains a tensin-like domain as well as a phosphatasecatalytic domain. PTEN negatively regulates the Akt/PKB signalingpathway functioning as a tumor suppressor. One or more diazo compoundsof this invention carrying a cell penetrating group can be employed toesterify PTEN to facilite cell uptake thereof. Employing the reversibleesterification reagents herein, esterified groups are removed after celluptake. In a specific embodiment, the cargo molecule is SCRIB, ascaffold protein which is involved in cell migration, cell polarity andcell proliferation [37]. One or more diazo compounds of this inventioncarrying a cell penetrating group, such as a fluorenyl, can be employedto esterify SCRIB to facilite cell uptake thereof. Employing thereversible esterification reagents herein, esterified groups are removedafter cell uptake to facilitate entry into the cytosol of the cell.

In specific embodiments exemplified herein, diazo compounds of thisinvention can be employed to esterify GFP (Green fluorescent protein)with one or more cell penetrating groups to facilitate cellular uptakeof the fluorescent protein. The diazo compounds herein can be employedwith various fluorescent proteins that are known in the art tofacilitate their uptake into cells.

The present invention provides a method of reversibly esterifying cargomolecules having one or more or two or more carboxylate groups forlabeling, or targeting and cellular uptake, wherein the ester groups areremovable by ester cleavage after cellular uptake.

Cellular uptake includes at least in part uptake into the cytosol. Inspecific embodiments, the method employs diazo compounds of formula I toreact with carboxylate groups on the cargo molecule to form esters.Preferably 2 or more carboxylate groups of the cargo molecule arereacted to covalently attach cell penetrating groups, for example viaester linkages. After esterification the cargo molecule is placed incontact with a cell or tissue and the esterified cargo molecule is takenup into the cell and at least in part into the cytosol. After uptakeinto the cell, the ester groups are removed within the cell, forexample, by the action of cellular enzymes (e.g., esterases).

All references throughout this application, for example patent documentsincluding issued or granted patents or equivalents; patent applicationpublications; and non-patent literature documents or other sourcematerial; are hereby incorporated by reference herein in theirentireties, as though individually incorporated by reference.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. References cited herein are incorporated byreference herein in their entirety to indicate the state of the art, insome cases as of their filing date, and it is intended that thisinformation can be employed herein, if needed, to exclude (for example,to disclaim) specific embodiments that are in the prior art. Forexample, when a compound is claimed, it should be understood thatcompounds known in the prior art, including certain compounds disclosedin the references disclosed herein (particularly in referenced patentdocuments), are not intended to be included in the claim.

When a group of substituents is disclosed herein, it is understood thatall individual members of those groups and all subgroups, including anyisomers and enantiomers of the group members, and classes of compoundsthat can be formed using the substituents are disclosed separately. Whena compound is claimed, it should be understood that compounds known inthe art including the compounds disclosed in the references disclosedherein are not intended to be included. When a Markush group or othergrouping is used herein, all individual members of the group and allcombinations and subcombinations possible of the group are intended tobe individually included in the disclosure.

Every formulation or combination of components described or exemplifiedcan be used to practice the invention, unless otherwise stated. Specificnames of compounds are intended to be exemplary, as it is known that oneof ordinary skill in the art can name the same compounds differently.When a compound is described herein such that a particular isomer orenantiomer of the compound is not specified, for example, in a formulaor in a chemical name, that description is intended to include eachisomers and enantiomer of the compound described individual or in anycombination.

One of ordinary skill in the art will appreciate that methods, deviceelements, starting materials, and synthetic methods other than thosespecifically exemplified can be employed in the practice of theinvention without resort to undue experimentation. All art-knownfunctional equivalents, of any such methods, device elements, startingmaterials, and synthetic methods are intended to be included in thisinvention. Whenever a range is given in the specification, for example,a temperature range, a time range, or a composition range, allintermediate ranges and subranges, as well as all individual valuesincluded in the ranges given are intended to be included in thedisclosure.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. Any recitation hereinof the term “comprising”, particularly in a description of components ofa composition or in a description of elements of a device, is understoodto encompass those compositions and methods consisting essentially ofand consisting of the recited components or elements. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, limitation or limitations which is notspecifically disclosed herein.

Without wishing to be bound by any particular theory, there can bediscussion herein of beliefs or understandings of underlying principlesrelating to the invention. It is recognized that regardless of theultimate correctness of any mechanistic explanation or hypothesis, anembodiment of the invention can nonetheless be operative and useful.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention.

THE EXAMPLES Example 1 General Experimental

Materials. Silica gel (40 μm; 230-400 mesh) was from SiliCycle. Reagentswere obtained from commercial sources and used without furtherpurification. Dichloromethane (DMC) and tetrahydrofuran were dried overa column of alumina. Thin-layer chromatography (TLC) was performed onplates of EMD 250 μm silica 60-F254.

Solvent removal. The phrase “concentrated under reduced pressure” refersto the removal of solvents and other volatile materials using a rotaryevaporator at water aspirator pressure (<20 torr) while maintaining awater bath below 40° C. Residual solvent was removed from samples athigh vacuum (<0.1 torr).

NMR spectroscopy. ¹H and ¹³C NMR spectra for all compounds were acquiredwith Bruker spectrometers in the National Magnetic Resonance Facility atMadison operating at 400, 500, 600, or 750 MHz. Chemical shift data arereported in units of δ□ppm) relative to an internal standard (residualsolvent or TMS).

Mass spectrometry. Electrospray ionization (ESI) mass spectrometry forsmall-molecule characterization was performed with a Micromass LCT atthe Mass Spectrometry Facility in the Department of Chemistry at theUniversity of Wisconsin-Madison. Matrix-assisted laserdesorption-ionization-time-of-flight (MALDI-TOF) mass spectrometry forprotein characterization was performed with a Voyager DE-Pro instrumentat the Biophysics Instrumentation Facility at the University ofWisconsin-Madison.

Abbreviations: AIBN (azobisisobutyroisonitrile); EtOAc (ethyl acetate);DCC (N, N′, dicyclohexylcarbodiimide); DBU (1,8-diazabicyclo[5.4.0]undec-7-ene); THF (tetrahydrofuran); MES (2-(N-morpholino)ethanesulfonicacid; DCM (dichloromethane).

Example 2 Synthesis And Characterization Data

Preparation of α-Bromoacid S1

4-Methoxyphenylacetic acid (5.000 g, 30.10 mmol) was dissolved in CCl₄(50 mL). N-Bromosuccinimide (NBS, 5.625 g, 31.6 mmol) and AIBN (0.985 g,6.0 mmol) were added. The resulting solution was heated to 80° C. andallowed to reflux overnight. The succinimide by-product was removed byfiltration, and the solution was concentrated under reduced pressure.The residue was purified by chromatography on silica gel, eluting with1:1 EtOAc/hexanes to afford S1 (5.705 g, 78%) as a white solid. Data forS1: ¹H NMR (500 MHz, CDCl₃, δ): 7.50 (d, 2H, J=8.8 Hz), 6.90 (d, 2H,J=8.8 Hz), 5.36 (s, 1H), 3.82 (s, 1H.) ¹³C NMR (125 MHz, CDCl₃, δ):173.4, 160.5, 130.2, 126.8, 114.3, 55.4, 45.9, HRMS (ESI⁻) m/z calcd forC₉H₉BrO₃ [M−H]⁻ 242.9662; found, 242.9660.

Preparation of α-Azido Acid S2

α-Bromo-4-methoxyphenylacetic acid 51 (0.802 g, 3.3 mmol) was dissolvedin 1:1 THF/H₂O (4 mL). Sodium azide (0.429 g, 6.6 mmol) was added, andthe resulting solution was stirred overnight. The solution was thenconcentrated under reduced pressure, and the residue was dissolved inEtOAc (50 mL). The resulting solution was washed with 0.1 M HCl (2×50mL). The organic layer was dried over anhydrous Na₂SO₄(s) andconcentrated under reduced pressure to afford S2 (0.412 g, 62%) as awhite solid.

Data for S2: ¹H NMR (500 MHz, CDCl₃, δ): 7.35 (d, 2H, J=8.7 Hz), 6.95(d, 2H, J=8.7 Hz), 5.00 (s, 1H), 3.83 (s, 3H). ¹³C NMR (125 MHz, CDCl₃,δ): 173.5, 160.5, 129.1, 125.2, 114.6, 64.6, 55.4, HRMS (ESI⁻) m/z calcdfor C₉H₉N₃O₃ [M−H]⁻ 206.0571; found, 206.0577.

Preparation of α-Azido 4-Methoxyphenylacetic Amide S3

α-Azido-4-methoxyphenylacetic acid S2 (0.412 g, 2.0 mmol) was dissolvedin THF (5 mL), and the resulting solution was cooled in an ice bath.N-Hydroxysuccinimide (NHS, 0.230 g, 2.0 mmol) was added, followed by theportion-wise addition of DCC (0.453 g, 2.2 mmol). The resulting solutionwas warmed to ambient temperature and stirred overnight. The slurry wasremoved by filtration, and the solution was concentrated under reducedpressure. The residue was dissolved in EtOAc (10 mL) and washed withsaturated aqueous NaHCO₃ (2×10 mL). The organic layer was dried overanhydrous Na₂SO₄(s) and concentrated under reduced pressure. The residuewas purified by chromatography on silica gel, eluting with 3:7EtOAc/hexanes, and used immediately. The NHS ester (0.4 g, 1.2 mmol) wasdissolved in CH₂Cl₂ (10 mL). Benzylamine (0.10 mL, 1.3 mmol) was addeddropwise, and the resulting solution was stirred overnight. The solutionwas then concentrated under reduced pressure. The residue was dissolvedin EtOAc (10 mL) and washed with 0.1 M HCI (2×10 mL) and saturatedaqueous NaHCO₃ (2×10 mL). The organic layer was dried over anhydrousanhydrous Na₂SO₄(s) and concentrated under reduced pressure to afford S3(0.255 g, 43%) as a white solid.

Data for S3: ¹H NMR (500 MHz, CD₃CN, δ):7.34-7.30 (m, 4H), 7.27-7.23 (m,3H), 6.97 (d, 2H, J=8.8 Hz), 4.99 (s, 1H), 4.37 (m, 2H), 3.80 (s, 3H).¹³C NMR (125 MHz, CD3CN, δ): 169.4, 161.0, 139.8, 130.2, 129.4, 128.4,128.2, 128.0, 115.1, 66.6, 55.9, 43.6. HRMS ESI⁺) m/z calcd forC₁₆H₁₆N₄O_(2 [)M+H]⁺ 297.1347; found, 297.1346.

Preparation of α-Diazo Amide 1

α-Azidoamide S3 (0.356 g, 1.2 mmol) was dissolved in 20:3 MeCN/H₂O (12mL), and the resulting solution was cooled in an ice bath.N-Succinimidyl 3-(diphenylphosphino)propionate (0.440 g, 1.24 mmol) wasadded slowly. The solution was warmed to ambient temperature and stirreduntil all azide was consumed (˜12 h as monitored by TLC). DBU (0.21 mL,1.4 mmol) was added, and the solution was stirred for 1 h. The solutionwas then diluted with brine (10 mL) and extracted with CH₂Cl₂ (2×20 mL).The organic layer was dried over anhydrous Na₂SO₄(s) and concentratedunder reduced pressure. The residue was purified by chromatography onsilica gel, eluting with 1:1 EtOAc/hexanes to afford 1 (0.095 g, 28%) asan orange solid.

Data for 1: ¹H NMR (500 MHz, CD₃CN, δ): 7.37 (d, 2H, J=8.9 Hz),7.34-7.29 (m, 4H), 7.26-7.23 (m, 1H), 4.43 (d, 2H, J=6.2 Hz), 3.80 (s,3H). ¹³C NMR (125 MHz, CDCl₃, δ): 165.4, 159.7, 138.4, 130.3, 128.7,127.7, 117.5, 115.3, 63.1, 55.4, 44.1. HRMS (ESI⁺) m/z calcd forC₁₆H₁₅N₃O₂ [M+H]⁺ 282.1238; found, 282.1232.

Preparation of α-Azido Acid S4

Imidazole-1-sulfonyl-azide hydrochloride was prepared as reportedpreviously. [29] Spectral data and yields match those reportedpreviously. α-Amino-4-methylphenylacetic acid (2.000 g, 12.1 mmol) wasdissolved in MeOH (24 mL). DBU (3.61 mL, 24.2 mmol), CuSO₄ (0.300 g, 1.2mmol), and azide (3.030 g, 14.5 mmol) were added sequentially. Theresulting solution was heated to 40° C. and stirred overnight. Thesolution was then concentrated under reduced pressure. The residue wasdissolved in EtOAc (30 mL) and washed twice with 1 M aqueous HCl (2×30mL). The organic layers were combined and dried over anhydrousNa₂SO₄(s). The solution was concentrated under reduced pressure. Theresidue was dissolved in benzene and recrystallized from benzene andhexanes to afford S4 (0.390 g, 17%) as a white solid.

Data for S4: ¹H NMR (600 MHz, CDCl₃, δ): 7.30 (d, 2H, J=8.1 Hz), 7.24(d, 2H, J=7.8 Hz), 5.01 (s, 1H), 2.37 (s, 3H). ¹³C NMR (150 MHz, CDCl₃,δ): 173.4, 139.7, 130.2, 129.9, 127.6, 64.9, 21.2. HRMS (ESI⁻) m/z calcdfor C₉H₉N₃O₂ [M−H]⁻ 190.0622; found, 190.0625.

Preparation of α-Azido-methylphenylacetic Amide S5

α-Azido 4-methylphenylacetic acid S4 (2.204 g, 11.6 mmol) was dissolvedin THF (30 mL) and cooled in an ice bath. N-Hydroxysuccinimide (1.334 g,11.6 mmol) was added, followed by portion-wise addition of DCC (2.637 g,12.8 mmol). The resulting solution was warmed to ambient temperature andstirred overnight. The slurry was removed by filtration, and thesolution was concentrated under reduced pressure. The residue wasdissolved in EtOAc (30 mL). The resulting solution was washed withsaturated aqueous NaHCO₃ (2×30 mL). The organic layer was dried overanhydrous Na₂SO₄(s), concentrated under reduced pressure, and usedimmediately. The NHS ester (2.5 g, 8.7 mmol) was dissolved in CH₂Cl₂ (30mL). Benzylamine (0.98 mL, 9.6 mmol) was added dropwise, and theresulting solution was stirred overnight. The solution was thenconcentrated under reduced pressure. The residue was dissolved in EtOAc(30 mL) and washed with 0.1 M HCI (2×30 mL) and saturated aqueous NaHCO₃(2×30 mL). The organic layer was dried over anhydrous anhydrousNa₂SO₄(s) and concentrated under reduced pressure to afford S5 (1.988 g,61%) as a white solid.

Data for S5: ¹H NMR (500 MHz, CD₃CN, δ): 7.33-7.28 (m, 4H), 7.26-7.22(m, 5H), 5.00 (s, 1H), 4.36 (dd, 2H, J=1.8, 6.2 Hz), 2.35 (s, 3H). ¹³CNMR (125 MHz, CD₃CN, 5): 169.2, 140.0, 139.8, 133.5, 130.4, 129.4,128.8, 128.0, 66.9, 43.6, 21.1. HRMS (ESI⁺) m/z calcd for C₁₆H₁₆N₄O[M+H]⁺ 281.1397; found, 281.1395.

Preparation of α-Diazo-methylphenylacetic Amide 2

α-Azido 4-methylphenylacetic amide S5 (1.995 g, 7.1 mmol) was dissolvedin 20:3 MeCN/H₂O (50 mL), and the resulting solution was cooled in anice bath. N-Succinimidyl 3-(diphenylphosphino)propionate (2.769 g, 7.8mmol) was added slowly. The solution was warmed to ambient temperatureand stirred until all azide was consumed (˜24 h as monitored by TLC).DBU (1.27 mL, 8.5 mmol) was added, and the solution stirred for 45 min.The solution was then diluted with brine (10 mL) and extracted withCH₂Cl₂ (2×30 mL). The organic layer was dried over anhydrous Na₂SO₄(s)and concentrated under reduced pressure. The residue was purified bychromatography on silica gel, eluting with 4:6 EtOAc/hexanes to afford 2(1.038 g, 55%) as an orange solid.

Data for 2: ¹H NMR (600 MHz, CD₃CN, δ): 7.33-7.23 (m, 9H), 6.63 (s, 1H),4.44 (d, 2H, J=6.2 Hz), 2.34 (s, 3H). ¹³C NMR (150 MHz, CD₃CN, δ):165.5, 140.7, 138.1, 130.9, 129.3, 128.2. 128.1, 127.9, 124.1, 63.74,44.0, 21.1. HRMS (ESI⁺) m/z calcd for C₁₆H₁₅N₃O [M+H]⁺ 266.1288; found,266.1292.

General Procedure for Preparation of Azides S6-S8

Each α-bromophenylacetic acid (23.3 mmol) was dissolved in a solution of1:1 THF/H₂O (24 mL). Sodium azide (1.512 g, 46.5 mmol) was added, andthe resulting solution was stirred overnight. The solution was thenconcentrated under reduced pressure. The residue was dissolved in EtOAc(50 mL), and washed with 0.1 M HCl (2×50 mL). The organic layer wasdried over anhydrous Na₂SO₄(s) and concentrated under reduced pressureto afford a white solid (S6: 4.076 g, 99%; S7: 4.016 g, 89%; S8: 3.761g, 77%).

Data for Azide S6: ¹H NMR (400 MHz, CDCl₃, δ): 7.43 (m, 5H), 5.05 (s,1H). ¹³C NMR (400 MHz, CDCl₃, δ): 174.0, 133.1, 129.6, 129.2, 127.7,65.1. HRMS (ESI⁺) m/z calcd for C₈H₇N₃O₂ [M+H]⁺ 177.0533; found,177.0538.

Data for Azide S7: ¹H NMR (400 MHz, CDCl₃, δ): 7.41 (dd, 2H, J=5.1, 8.5Hz), 7.12 (t, 2H, J=8.4 Hz), 5.05 (s, 1H). ¹³C NMR (100 MHz, CDCl₃, δ):175.0, 163.5 (d, J=249.6 Hz), 129.8 (d, J=8.5 Hz) 129.1 (d, J=2.6 Hz),116.5 (d, J=22.1 Hz), 64.5.HRMS (ESI⁻) m/z calcd for C₈H₆FN₃O₂ [M−H]⁻194.0371; found, 194.0378.

Data for Azide S8: ¹H NMR (400 MHz, CDCl₃, δ): 7.41 (d, 2H, J=8.4 Hz),7.37 (d, 2H, J=8.3 Hz), 5.06 (s, 1H). ¹³C NMR (125 MHz, CDCl₃, δ):174.7, 135.8, 131.5, 129.5, 129.0, 64.3. HRMS (ESI⁻) m/z calcd forC₈H₆ClN₃O₂ [M−H]⁻ 210.0075; found, 210.0078.

General Procedure for Preparation of Amides S9-S11

Each α-azidoacetic acid (S6-S8) (15.4 mmol) was dissolved in THF (30mL), and the resulting solution was cooled in an ice bath.N-Hydroxysuccinimide (NHS) (1.772 g, 15.4 mmol) was added, followed byportion-wise addition of DCC (3.177 g, 15.4 mmol). The solution waswarmed to ambient temperature and stirred overnight. The slurry wasremoved by filtration, and the solution was concentrated under reducedpressure. The residue was dissolved in EtOAc (50 mL) and washed withsaturated aqueous NaHCO₃ (2×50 mL). The organic layer was dried overanhydrous Na₂SO₄(s) and concentrated under reduced pressure. The residuewas purified by chromatography on silica gel, eluting with 1:1EtOAc/hexanes. The resulting solution was then concentrated underreduced pressure and used immediately. The NHS ester (10.5 mmol) wasdissolved in CH₂Cl₂ (105 mL). Benzylamine (1.16 mL, 10.6 mmol) was addeddrop-wise, and the resulting solution was stirred overnight. Thesolution was concentrated under reduced pressure. The residue wasdissolved in EtOAc (50 mL) and washed with 0.1 M HCl (2×50 mL) andsaturated aqueous NaHCO₃ (2×50 mL). The organic layer was dried overanhydrous Na₂SO₄(s) and concentrated under reduced pressure. The residuewas purified by chromatography on silica gel, eluting with 30%EtOAc/hexanes to afford a white solid (S9: 2.384 g, 58% for 2 steps;S10: 2.062 g, 47% for 2 steps; S11: 2.179 g, 47% for 2 steps).

Data for Amide S9: ¹H NMR (500 MHz, CD₃CN, δ): 7.43-7.42 (m, 5H),7.31-7.29 (m, 2H), 7.26-7.22 (m, 3H), 5.06 (s, 1H), 4.37 (d, 2H, J=6.2).¹³C NMR (125 MHz, CDCl₃, δ): 167.8, 137.5, 134.9, 129.2, 129.1, 128.8,127.8, 127.73, 127.67, 67.4, 43.7. HRMS (ESI⁺) m/z calcd for C₁₅H₁₄N₄O[M+H]⁺ 267.1241; found, 267.1241.

Data for Amide S10: ¹H NMR (600 MHz, CD₃CN, δ): 7.45-7.42 (dd, 2H,J=5.4, 8.7 Hz), 7.23-7.30 (m, 2H), 7.26-7.22 (m, 3H), 7.18-7.15 (m, 2H),5.08 (s, 1H), 4.37 (dd, 2H, J=3.0, 6.2 Hz). ¹³C NMR (100 MHz, CDCl₃, δ):167.6, 163.1 (d, J=249.2 Hz), 137.5, 130.9 (d, J=2.0 Hz), 129.5 (d,J=8.5 Hz), 128.8, 127.8, 116.2 (d, J=21.8 Hz), 105.0, 66.6, 43.7. HRMS(ESI⁺) m/z calcd for C₁₅H₁₃FN₄O [M+H]⁺ 285.1147; found, 285.1150.

Data for Amide S11: ¹H NMR (500 MHz, CD₃CN, δ): 7.44-7.39 (m, 4H),7.33-7.27 (m, 2H), 7.25-7.22 (m, 3H), 5.08 (s, 1H), 4.36 (m, 2H). ¹³CNMR (125 MHz, CD₃CN, 5): 168.8, 139.7, 135.5, 135.2, 130.4, 129.9,129.4, 128.2, 128.0, 66.3, 43.6. HRMS (ESI⁺) m/z calcd for C₁₅H₁₃ClN₄O[M+H]⁺ 301.0851; found, 301.0850.

General Procedure for Preparation of Diazo Compounds 3-5

Each α-azidobenzylamide (S9-S11) (7.3 mmol) was dissolved in a solutionof 20:3 THF:H₂O (75 mL) and cooled in an ice bath. N-Succinimidyl3-(diphenylphosphino)propionate (2.734 g, 7.7 mmol) was added slowly.The resulting solution was warmed to ambient temperature and stirreduntil all azide was consumed (6-12 h as monitored by TLC). Saturatedaqueous NaHCO₃ (73 mL) was added, and the solution was stirredovernight. The solution was then diluted with brine (50 mL) andextracted with CH₂Cl₂ (2×70 mL). The organic layer was dried overanhydrous Na₂SO₄(s) and concentrated under reduced pressure. The residuewas purified by chromatography on silica gel, eluting with 1:1EtOAc/hexanes to afford an orange solid (3: 1.012 g, 55%; 4: 0.887 g,45%; 5: 0.877 g, 42%).

Data for Diazo 3: ¹H NMR (600 MHz, CD₃CN, δ): 7.46-7.41 (m, 4H),7.34-7.28 (m, 4H), 7.28-7.23 (m, 2H), 6.73 (s, 1H), 4.44 (d, 2H, J=6.1Hz). ¹³C NMR (125 MHz, CD₃CN, δ): 165.1, 140.6, 130.2, 129.3, 128.2,127.8, 127.7, 127.6, 127.4, 64.0, 43.9. HRMS (ESI⁺) m/z calcd forC₁₅H₁₃N₃O [M+H]⁺ 252.1132; found, 252.1125.

Data for Diazo 4: ¹H NMR (500 MHz, CD₃CN, δ): 7.49-7.46 (dd, 2H, J=5.4,8.6 Hz), 7.34-7.29 (m, 4H), 7.26-7.23 (m, 1H), 7.20-7.16 (t, 2H, J=8.8),6.70 (s, 1H), 4.43 (d, 2H, J=6.2). ¹³C NMR (125 MHz, CD₃CN, δ): 165.2,162.5 (d, J=244.9 Hz), 140.6, 130.2 (d, J=8.3 Hz), 129.2, 128.1, 127.8,123.4 (d, J=3.1 Hz), 116.9 (d, J =22.1 Hz), 62.99, 43.8. HRMS (ESI⁺) m/zcalcd for C₁₅H₁₂FN₃O [M+H]⁺ 270.1038; found, 270.1032.

Data for Diazo 5: ¹H NMR (500 MHz, CD₃CN, δ): 7.45 (d, 2H, J=8.8 Hz),7.42 (d, 2H, 8.9 Hz), 7.35-7.30 (m, 4H), 7.28-7.26 (m, 1H), 6.79 (s,1H), 4.44 (d, 2H, J=6.1 Hz). ¹³C NMR (125 MHz, CDCl₃, δ): 164.1, 138.1,133.5, 129.9, 128.8, 128.5, 127.8, 127.7,124.7, 63.5, 44.2. HRMS (ESI⁺)m/z calcd for C₁₅H₁₂ClN₃O [M+H]⁺ 286.0742; found, 286.0748.

Preparation of Ester S12

4-(Trifluoromethyl)phenylacetic acid (5.000 g, 24.5 mmol) was dissolvedin THF (50 mL), and the resulting solution was cooled in an ice bath.N-Hydroxysuccinimide (2.818 g, 24.5 mmol) was added, followed by DCC(5.047 g, 24.5 mmol). The solution was warmed to ambient temperature andstirred overnight. The slurry was removed by filtration, and thesolution was concentrated under reduced pressure. The residue wasdissolved in EtOAc (50 mL) and washed with saturated aqueous NaHCO₃(2×50 mL). The organic layer was dried over anhydrous Na₂SO₄(s) andconcentrated under reduced pressure. The residue was purified bychromatography on silica gel, eluting with 1:1 EtOAc/hexanes to affordS12 (7.301 g, 99%) as a white solid.

Data for Ester S12: ¹H NMR (400 MHz, CDCl₃, δ): 7.63 (d, 2H, J=7.99 Hz),7.48 (d, 2H, J=7.92 Hz), 4.00 (s, 2H), 2.84 (s, 4H). ¹³C NMR (125 MHz,CDCl₃, δ): 168.9, 166.1, 135.27, 130.2 (q, J=32.6 Hz), 129.7, 125.8 (q,J=3.7 Hz), 123.9 (q, J=272.1 Hz), 37.4, 25.6. HRMS (EI⁺) m/z calcd forC₁₃H₁₀F₃NO₄ [M+H]⁺ 301.0557; found, 301.0565.

Preparation of α-Bromoester S13

Ester S12 (3.763 g, 12.5 mmol) was dissolved in CCl₄ (25 mL).N-Bromosuccinimide (3.329 g, 18.7 mmol) and AIBN (0.394 g, 2.4 mmol)were added. The resulting solution was heated to 80° C. and allowed toreflux overnight. The succinimide by-product was removed by filtration,and solution was concentrated under reduced pressure. The residue waspurified by chromatography on silica gel, eluting with 1:1 EtOAc/hexanesto afford S13 (2.037 g, 43%) as a white solid.

Data for S13: ¹H NMR (500 MHz, CDCl₃, δ): 7.72 (d, 2H, J=8.3 Hz), 7.69(d, 2H, J=8.6 Hz), 5.68 (s, 1H), 2.86 (s, 4H). ¹³C NMR (125 MHz, CDCl₃,δ): 168.2, 163.8, 137.7, 131.9 (q, J=32.8 Hz), 129.2, 126.1 (q, J=3.7Hz), 123.6 (q, J=272.5 Hz), 40.7, 25.6. HRMS (EI⁺) m/z calcd forC₁₃H₉BrF₃NO₄ [M+H]⁺ 378.9662; found, 378.9667.

Preparation of α-Bromoamide S14

α-Bromoester S13 (3.297 g, 8.7 mmol) was dissolved in CH₂Cl₂ (80 mL).Benzylamine (0.91 mL, 8.7 mmol) was added drop-wise, and the resultingsolution was stirred overnight. The solution was concentrated underreduced pressure, and the residue was dissolved in EtOAc (50 mL). Thesolution was washed with 0.1 M HCl (2×50 mL) and saturated aqueousNaHCO₃ (2×50 mL). The organic layers were dried over anhydrous Na₂SO₄(s)and concentrated under reduced pressure. The residue was purified withchromatography on silica gel, eluting with 1:1 EtOAc/hexanes to affordS14 (1.456 g, 45%) as a white solid.

Data for S14: ¹H NMR (500 MHz, CD₃CN, δ): 7.76 (d, 2H, J=8.3 Hz), 7.72(d, 2H, J=2H), 7.51 (s, 1H), 7.35 (t, 3H, J=7.4 Hz), 7.29 (t, 3H, J=7.7Hz), 5.59 (s, 1H), 4.40 (m, 2H). ¹³C NMR (125 MHz, CDCl₃, δ): 166.2,141.2, 137.1, 131.1 (q, J=32.8 Hz), 128.9, 128.8, 128.0, 127.8, 125.9(q, J=3.7 Hz), 123.7 (q, J=272.3 Hz), 49.8, 44.6. HRMS (ESI⁺) m/z calcdfor C₁₆H₁₃BrF₃NO [M+H]⁺ 372.0206; found, 372.0210.

Preparation of α-Azidoamide S15

α-Bromoamide S14 (1.823 g, 4.9 mmol) was dissolved in 1:1 THF/H₂O.Sodium azide (0.637 g, 9.8 mmol) was added, and the resulting solutionwas stirred overnight. The solution was concentrated under reducedpressure. The residue was dissolved in EtOAc (50 mL), and the resultingsolution was washed twice with 0.1 M HCl (2×50 mL). The organic layerwas dried over anhydrous Na₂SO₄(s) and concentrated under reducedpressure to afford S15 (1.018 g, 62%) as a white solid.

Data for S15: ¹H NMR (500 MHz, CD₃CN, δ): 7.74 (d, 2H, J=8.1 Hz), 7.60(d, 2H, J=8.0 Hz), 7.42 (s, 1H), 7.31 (m, 2H), 7.24 (m, 3H), 5.19 (s,1H), 4.37 (d, 2H, J=6.2 Hz). ¹³C NMR (125 MHz, CD₃CN, δ): 170.2, 142.8,141.4, 132.9 (q, J=32.3 Hz), 131.2, 131.1, 130.0, 129.8, 128.5 (q, J=3.9Hz), 126.9 (q, J=271.3 Hz), 68.2, 45.4. HRMS (ESI⁺) m/z calcd for(C₁₆H₁₃F₃N₄O) [M+H]⁺ 335.1115; found, 335.1112.

Preparation of α-Diazoamide 6

α-Azidoamide S15 (1.002 g, 2.99 mmol) was dissolved in 20:3 THF/H₂O (30mL), and the resulting solution was cooled in an ice bath.N-Succinimidyl 3-(diphenylphosphino)propionate (1.115 g, 3.14 mmol) wasadded slowly. The solution was warmed to ambient temperature and stirreduntil all azide was consumed (˜5 h as monitored by TLC). Saturatedaqueous NaHCO₃ (30 mL) was added, and the solution was stirredovernight. The solution was diluted with brine (30 mL) and extractedwith CH₂Cl₂ (2×30 mL). The organic layer was dried over anhydrousNa₂SO₄(s) and concentrated under reduced pressure. The residue waspurified by chromatography on silica gel, eluting with 1:1 EtOAc/hexanesto afford 6 (0.382 g, 40%) as an orange solid.

Data for 6: ¹H NMR (400 MHz, CDCl₃, δ): 7.65 (d, 2H, J=8.0 Hz), 7.50 (d,2H, J=8.1 Hz), 7.38-7.31 (m, 5H), 5.70 (s, 1H), 4.59 (d, 2H, J=4.6 Hz).¹³C NMR (125 MHz, CD₃CN, δ): 164.2, 140.4, 132.9, 128.3, 127.9, 127.6(q, J=32.4 Hz), 126.5 (q, J=3.9 Hz), 126.3, 125.3 (q, J=270.8 Hz), 64.0,43.9. HRMS (ESI⁺) m/z calcd for C₁₆H₁₂F₃N₃O [M+H]⁺ 320.1006; found,320.0993.

Example 3 Measurement of Reaction Rate Constants

Each diazo compound and BocGlyOH were dissolved separately in CD₃CN at aconcentration of 50 mM. The solutions were combined in an NMR tube at anequimolar ratio, mixed, and then inserted immediately into an NMRspectrometer. A 16-scan ¹H NMR spectrum was acquired every 10 min.Percent conversion was monitored by disappearance of starting materialand appearance of product as determined by integration of multiple ¹HNMR spectral peaks. No other products were apparent by ¹H NMRspectroscopy. The value of the second-order rate constant was determinedby linear regression analysis of a plot of 1/[diazo] versus time (datanot shown). All reactions were performed in triplicate.

Example 4 Esterification of BocGlyOH

Diazo compound 1 (0.005 g, 0.02 mmol) and BocGlyOH (0.003 g, 0.02 mmol)were added to a 1:1 solution of acetonitrile/100 mM MES-HCl buffer at pH5.5, and the resulting solution was stirred for 6 h at ambienttemperature. The reaction mixture was concentrated under reducedpressure, and the ratio of products was determined by integration of ¹HNMR spectral peaks.

Data for S16: ¹H NMR (400 MHz, CD₃CN, δ): 7.60 (s, 1H), 7.37-7.22 (m,7H), 6.93 (d, 2H, J=8.4 Hz), 5.91 (s, 1H), 5.74 (s, 1H), 4.43-4.31 (m,2H), 3.94-3.82 (m, 2H), 3.79 (s, 3H), 1.38 (s, 9H). ¹³C NMR (100 MHz,CD₃CN, δ): 170.4, 169.3, 161.1, 157.4, 139.9, 129.9, 129.3, 128.6,128.1, 127.9, 114.8, 80.3, 76.7, 55.9, 43.2, 28.4 HRMS (ESI⁺) m/z calcdfor C₂₃H₂₈N₂O₆ [M+H]⁺ 429.2021; found, 429.2021.

Data for S17: ¹H NMR (500 MHz, CD3CN, 5): 7.47 (s, 1H), 7.33-7.25 (m,4H), 7.23-7.21 (m, 3H), 6.90 (d, 2H, J=8.8 Hz), 4.97 (d, 1H, J=4.5 Hz),4.40-4.32 (m, 2H), 4.16 (d, 2H, J=4.5 Hz), 3.78 (s, 3H). ¹³C NMR (125MHz, CD₃CN, δ): 173.3, 160.4, 140.3, 133.8, 129.3, 129.0, 128.1, 127.8,114.5, 74.3, 55.8, 43.1. HRMS (ESI⁺) m/z calcd for C₁₆H₁₇NO₃ [M+H]⁺272.1282; found, 272.1278.

Diazo compound 2 (0.005 g, 0.02 mmol) and BocGlyOH (0.003 g, 0.02 mmol)were added to 1:1 acetonitrile/100 mM MES-HCl buffer at pH 5.5, and theresulting solution was stirred for 6 h at ambient temperature. Thesolution was then concentrated under reduced pressure, and the ratio ofproducts was determined by integration of ¹H NMR spectral peaks.

Data for S18: ¹H NMR (500 MHz, CD₃CN, δ): 7.65 (s, 1H), 7.33-7.28 (m,4H), 7.25-7.20 (m, 5H), 5.92 (s, 1H), 5.77 (s, 1H), 4.42-4.31 (m, 2H),3.92-3.82 (m, 2H), 2.34 (s, 3H), 1.38 (s, 9H). ¹³C NMR (125 MHz, CD₃CN,δ):170.4, 169.2, 157.4, 140.0, 139.8, 133.7, 130.1, 129.3, 128.3, 128.1,127.9, 80.3, 76.8, 43.2, 43.2, 28.4, 21.2. HRMS (ESI⁺) m/z calcd forC₂₃H₂₈N₂O₅ [M+NH₄]⁺ 430.2337; found, 430.2336.

Data for S19: ¹H NMR (500 MHz, CD₃CN, δ): 7.46 (s, 1H), 7.31-7.28 (m,4H), 7.25-7.21 (m, 3H), 7.17 (d, 2H, J=7.9 Hz), 4.99 (d, 1H, J=4.2 Hz),4.40-4.32 (m, 2H), 4.18 (d, 1H), J=4.5 Hz), 2.32 (s, 1H). ¹³C NMR (125MHz, CD₃CN, δ): 173.3, 140.3, 138.74, 138.71, 129.8, 129.3, 128.1,127.9, 127.6, 74.6, 43.1, 21.1. HRMS (ESI⁺) m/z calcd for C₁₆H₁₇NO₂[M+H]⁺ 256.1333; found, 256.1330.

Diazo compound 3 (0.005 g, 0.02 mmol) and BocGlyOH (0.004 g, 0.02 mmol)were added to 1:1 acetonitrile/100 mM MES-HCl buffer at pH 5.5, and theresulting solution was stirred for 6 h at ambient temperature. Thereaction mixture was then concentrated under reduced pressure, and theratio of products was determined by integration of ¹H NMR spectralpeaks.

Data for S20: ¹H NMR (750 MHz, CD₃CN, δ): 7.65 (s, 1H), 7.46 (m, 2H),7.40 (m, 3H), 7.30 (t, 2H, J=7.4 Hz), 7.23 (m, 3H), 5.99 (s, 1H), 5.78(s, 1H), 4.41 (dd, 1H, J=6.3, 15.2 Hz), 4.35 (dd, 1H, J=6.1, 15.2 Hz),3.92 (dd, 1H, J=6.2, 17.9 Hz), 3.88 (dd, 1H, J=5.7, 18.0 Hz), 1.40 (s,9H). ¹³C NMR (125 MHz, CDCl₃, δ):168.7, 168.0, 156.4, 137.9, 135.0,129.1, 128.8, 128.6, 127.8, 127.5, 127.4, 80.6, 76.2, 43.4, 43.0, 28.2.HRMS (ESI⁺) m/z calcd for C₂₂H₂₆N₂O₅ [M+H]⁺ 399.1915; found, 399.1917.

Data for S21: ¹H NMR (750 MHz, CD₃CN, δ): 7.48 (s, 1H), 7.43 (d, 2H,J=7.4 Hz), 7.36 (t, 2H, J=7.4 Hz), 7.31 (m, 3H), 7.24 (m, 3H), 5.04 (d,1H, J=2.8 Hz), 4.37 (m, 2H), 4.28 (d, 1H, J=3.8 Hz). ¹³C NMR (125 MHz,CD₃CN, δ):173.1, 141.6, 140.3, 129.3, 129.2, 128.8, 128.1, 127.9, 127.6,74.7, 43.1. HRMS (ESI⁺) m/z calcd for C₁₅H₁₅NO₂ [M+H]⁺ 242.1176; found,242.1169.

Diazo 4 (0.005 g, 0.02 mmol) and BocGlyOH (0.003 g, 0.02 mmol) wereadded to 1:1 acetonitrile/100 mM MES-HCl buffer at pH 5.5, and theresulting solution was stirred for 6 h at ambient temperature. Thereaction mixture was then concentrated under reduced pressure, and theratio of products was determined by integration of ¹H NMR spectralpeaks.

Data for S22: ¹H NMR (500 MHz, CD₃CN, δ): □7.66 (s, 1H), 7.48 (dd, 2H,J=5.4, 8.6 Hz), 7.30 (t, 2H, J=7.3 Hz), 7.25-7.20 (m, 3H), 7.14 (t, 2H,J=8.9 Hz), 5.97 (s, 1H), 5.77 (s, 1H), 4.40 (dd, 1H, J=6.3, 15.2 Hz),4.34 (dd, 1H, J=6.1, 15.2 Hz), 3.94-3.84 (m, 2H), 1.38 (s, 9H). ¹³C NMR(125 MHz, CDCl₃, δ):168.6, 167,9, 163.1 (d, J=248.2 Hz), 156.4, 137.8,131.0 (d, J=3.3 Hz), 129.4 (d, J=8.5 Hz), 127.8, 127.5, 115.8 (d, J=21.8Hz), 80.7, 75.5, 43.4, 43.0, 28.2. HRMS (ESI⁺) m/z calcd for C₂₂H₂₅FN₂O₅[M+H]⁺ 417.1821; found, 417.1816.

Data for S23: ¹H NMR (400 MHz, CD₃CN, δ): 7.53 (s, 1H), 7.45-7.42 (m,2H), 7.32-7.28 (m, 2H), 7.24-7.20 (m, 3H), 7.09 (t, 2H, J=8.9 Hz), 5.04(s, 1H), 4.41-4.31 (m, 2H). ¹³C NMR (125 MHz, CD₃CN, δ): 174.7, 165.0(d, J=243.7 Hz), 142.0, 139.6, 131.3 (d, J=8.3 Hz), 131.1., 129.8,129.6, 117.6 (d, J=21.7 Hz), 75.7, 44.8. HRMS (ESI⁺) m/z calcd forC₁₅H₁₄FNO₂ [M+H]⁺ 260.1082; found, 260.1080.

Diazo 5 (0.005 g, 0.02 mmol) and BocGlyOH (0.003 g, 0.02 mmol) wereadded to 1:1 acetonitrile/100 mM MES-HCl buffer at pH 5.5, and theresulting solution was stirred for 6 h at ambient temperature. Thereaction mixture was then concentrated under reduced pressure, and theratio of products was determined by integration of ¹H NMR spectralpeaks.

Data for S24: ¹H NMR (500 MHz, CD₃CN, δ): 7.61 (s, 1H), 7.45-7.40 (m,4H), 7.31-7.29 (m, 2H), 7.25-7.21 (m, 3H), 5.98 (s, 1H), 5.74 (s, 1H),4.42-4.32 (m, 2H), 3.90 (m, 2H), 1.39 (s, 9H). ¹³C NMR (125 MHz, CDCl₃,δ): 168.5, 167.6, 156.4, 137.7, 135.1, 135.6, 128.9, 128.8, 128.6,127.8, 127.5, 80.8, 75.4, 43.4, 43.0, 28.2. HRMS (ESI⁺) m/z calcd forC₂₂H₂₅ClN₂O₅ [M+NH₄]⁺ 450.1791; found, 450.1785.

Data for S25: ¹H NMR (500 MHz, CD₃CN, δ): 7.47 (s, 1H), 7.42 (d, 2H,J=8.5 Hz), 7.37 (d, 2H, 8.6 Hz), 7.32-7.29 (m, 2H), 7.25-7.21 (m, 3H),5.04 (d, 1H, J=1.8 Hz), 4.36 (m, 2H), 4.31 (d, 1H, J=3.4 Hz). ¹³C NMR(125 MHz, CD₃CN, δ): 172.7, 140.5, 140.2, 134.0, 129.3, 129.21, 129.18,128.1, 127.9, 73.9, 43.1. HRMS (ESI⁺) m/z calcd for C₁₅H₁₄ClNO₂ [M+H]⁺276.0786; found, 276.0789.

Diazo 6 (0.005 g, 0.02 mmol) and BocGlyOH (0.003 g, 0.02 mmol) wereadded to 1:1 acetonitrile/100 mM MES-HCl buffer at pH 5.5, and theresulting solution was stirred for 6 h at ambient temperature. Thereaction mixture was then concentrated under reduced pressure, and theratio of products was determined by integration of ¹H NMR spectralpeaks.

Data for S26: ¹H NMR (500 MHz, CD₃CN, δ): 7.73-7.71 (m, 3H), 7.65 (d,2H, J=8.3 Hz), 7.31-7.28 (m, 2H), 7.25-7.20 (m, 3H), 6.06 (s, 1H), 5.77(s, 1H), 4.42-4.32 (m, 2H), 3.97-3.87 (m, 2H), 1.38 (s, 1H). ¹³C NMR(125 MHz, CD₃CN, δ): 170.3, 168.4, 157.4, 141.1, 139.7, 131.1 (q, J=32.4Hz), 129.4, 128.8, 128.1, 128.0, 126.3 (q, J=3.9 Hz), 125.1 (q, J=271.3Hz), 80.4, 76.1, 43.4, 43.2, 28.4. HRMS (ESI⁺) m/z calcd forC₂₃H₂₅F₃N₂O₅ [M+NH_(4]) ⁺ 484.2037; found, 484.2054.

Data for S27: ¹H NMR (400 MHz, CD₃CN, δ): 7.69-7.62 (m, 4H), 7.56 (s,1H), 7.31-7.20 (m, 5H), 5.54 (s, 1H), 5.14 (d, 1H, J=4.6 Hz), 4.45 (d,1H, J=4.8 Hz), 4.37-4.35 (m, 2H). ¹³C NMR (125 MHz, CD₃CN, δ): 172.3,146.0, 140.1, 130.1 (q, J=32.3 Hz), 129.3, 128.1, 128.9, 126.2 (q,J=41.3 Hz), 125.3 (q, J=271.3 Hz), 74.0, 43.1. HRMS calcd for(C₁₆H₁₄F₃NO₂) [M+H]⁺ 310.1050; found, 310.1043.

Example 5 Esterification of Other Small Molecules

Diazo compound 2 (0.005 g, 0.02 mmol) and BocSerOH (0.004 g, 0.02 mmol)were added to 1:1 acetonitrile/100 mM MES-HCl buffer at pH 5.5, and theresulting solution was stirred for 6 h at ambient temperature. Thesolution was then concentrated under reduced pressure, and the ratio ofproducts was determined by integration of ¹H NMR spectral peaks. Datafor S19 are reported above; data for S28 are reported below (bothdiastereomers). No other products were observed by TLC or ¹H NMRspectroscopy.

Data for S28: ¹H NMR (500 MHz, CD₃CN, Diastereomer A, 5): 7.72 (s, 1H),7.35 (d, 2H, J=8.0 Hz), 7.30 (t, 2H, J=7.3 Hz), 7.24 (t, 3H, J=7.7 Hz),7.18 (d, 2H, J=7.2 Hz), 5.96 (s, 1H), 5.79 (d, 1H, J=6.8 Hz), 4.38-4.33(m, 2H), 4.32-4.29 (m, 1H), 4.08-4.03 (m, 1H), 3.77-3.69 (m, 2H), 2.34(s, 3H), 1.40 (s, 9H). ¹H NMR (500 MHz, CD₃CN, Diastereomer B, δ): 7.64(s, 1H), 7.36-7.28 (m, 4H), 7.25-7.17 (m, 5H), 5.95 (s, 1H), 5.84 (d,1H, J=7.8 Hz), 4.41-4.30 (m, 2H), 4.28-4.25 (m, 1H), 3.86-3.82 (m, 1H),3.79-3.72 (m, 1H), 3.41 (t, 3H, J=5.7 Hz), 2.34 (s, 3H), 1.36 (s, 9H).¹³C NMR (125 MHz, CD₃CN, Diasteromer A, δ): 171.3, 169.7, 157.0, 140.2,139.6, 133.2, 130.2, 129.3, 128.5, 128.1, 128.0, 80.3, 77.0, 63.3, 57.1,43.4, 28.4, 21.2. ¹³C NMR (125 MHz, CD₃CN, Diastereomer B, δ): 171.2,169.3, 156.7, 139.9, 139.8, 133.6, 130.1, 129.3, 128.4, 128.1, 127.9,80.3, 77.0, 62.8, 57.1, 43.3, 28.4, 21.1. HRMS (ESI⁺) m/z calcd forC₂₄H₃₀N₂O₆ [M+H]⁺ 443.2177; found, 443.2185 (Diastereomer A), 443.2183(Diastereomer B).

Diazo compound 2 (0.005 g, 0.02 mmol) and p-hydroxybenzoic acid (0.003g, 0.02 mmol) were added to 1:1 acetonitrile/100 mM MES-HCl buffer at pH5.5, and the resulting solution was stirred for 6 h at ambienttemperature. The solution was then concentrated under reduced pressure,and the ratio of products was determined by integration of ¹H NMRspectral peaks. Data for S19 are reported above; data for S29 arereported below. No other products were observed by TLC or ¹H NMRspectroscopy.

Data for S29: ¹H NMR (500 MHz, CD3CN, δ): 7.98 (d, 2H, J=8.8 Hz), 7.76(s, 1H), 7.44 (d, 2H, J=8.1 Hz), 7.39 (s, 1H), 7.29-7.18 (m, 7H), 6.89(d, 2H, J=8.8 Hz), 6.06 (s, 1H), 4.36 (d, 2H, J=6.2 Hz), 2.35 (s, 3H).¹³C NMR (125 MHz, CD₃CN, δ): 169.7, 165.8, 162.6, 140.0, 139.8, 134.2,133.0, 130.1, 129.3, 128.3, 128.0, 127.9, 121.9, 116.1, 76.8, 43.1,21.2.HRMS (ESI⁺) m/z calcd for C₂₃H₂₁NO₄ [M+H]⁺ 376.1544; found,376.1539.

Diazo compound 2 (0.005 g, 0.02 mmol) and 3-mercaptopropanoic acid(0.002 g, 0.02 mmol) were added to 1:1 acetonitrile/100 mM MES-HClbuffer at pH 5.5, and the resulting solution was stirred for 6 h atambient temperature. The solution was then concentrated under reducedpressure, and the ratio of products was determined by integration of ¹HNMR spectral peaks. Data for S19 are reported above; data for S30 arereported below. No other products were observed by TLC or ¹H NMRspectroscopy.

Data for S30: ¹H NMR (500 MHz, CD₃CN, δ): 7.38 (s, 1H), 7.34 (d, 2H,J=8.1 Hz), 7.29 (t, 2H, J=7.3 Hz), 7.25-7.19 (m, 5H), 5.91 (s, 1H), 4.35(d, 2H, J=6.2 Hz), 2.80-2.70 (m, 4H), 2.34 (s, 3H), 1.89 (t, 1H, J=8.2Hz). ¹³C NMR (125 MHz, CD₃CN, δ): 171.5, 169.4, 139.9, 139.8, 133.9,130.1, 129.3, 128.3, 128.1, 127.9, 76.6, 43.1, 39.1, 21.1, 20.2. HRMS(ESI⁺) m/z calcd for (C₁₉H₂₁NO₃S) [M+H]⁺ 344.1315; found, 344.1315.

Example 6 Protein Labeling

where n indicates the number of esters formed in the protein.

9-Diazofluorene was prepared as described previously. [5] Yields andspectra matched the published data. Ribonuclease A (0.010 g, 0.73 μmol)was dissolved in 1 mL of 10 mM MES-HCl buffer at pH 5.5. 9-Diazofluorene(0.007 g, 0.036 mmol) was dissolved in 5 mL of CH₃CN. A 100-μL aliquotof the diazo stock solution was added to a 100-μL aliquot of the RNase Astock solution. The resulting mixture was mixed by nutation for 4 h at37° C. Any remaining diazo compound was then quenched by addition of 10μL of 17.4 M acetic acid. Acetonitrile was removed by concentrationunder reduced pressure, and the aqueous solution of labeled protein wasanalyzed by MALDI-TOF mass spectrometry (FIGS. 5A-B).

where n indicates the number of esters formed in the protein.

Ribonuclease A (0.010 g, 0.73 μmol) was dissolved in 1 mL of 10 mMMES-HCl buffer at pH 5.5. Diazo compound 2 (0.095 g, 0.036 mmol) wasdissolved in 5 mL of CH₃CN. A 100-μL aliquot of the diazo stock solutionwas added to a 100-μL aliquot of the RNase A stock solution. Theresulting mixture was mixed by nutation for 4 h at 37° C. Any remainingdiazo compound was then quenched by addition of 10 μL of 17.4 M aceticacid. Acetonitrile was removed by concentration under reduced pressure,and the aqueous solution of labeled protein was analyzed by MALDI-TOFmass spectrometry (FIGS. 5A-B).

Example 7 Protein Labeling

Angiogenin is used as a model protein to test the efficiency andreversibility of labeling. Treatment of angiogenin with a stoichiometricamount of a diazo-compound of formula 1, particularly compounds 2, 3 and4 results in the addition of up to 6 labels as determined by MALDI-TOFmass spectrometry (data not shown). Labeled protein is treated with HeLacell extract, which completely removed all labels demonstratingbioreversibility of labeling.

In a specific example, a stock solution of diazo 3 (19.1 mg, 76 μmol)was prepared by dissolving diazo 3 in 2 mL MeCN. A 200 μL portion ofstock solution was added to 200 μL of FLAG-angiogenin (2.9 mg/mL in 10mM Bis-Tris buffer, pH 6.0). The resulting mixture was mutated for 12hours at 25° C. The extent of labeling was determined by MALDI-TOF massspectrometry (data not shown).

A stock solution of diazo 4 (20.4 mg, 76 μmol) was prepared bydissolving diazo 4 in 2 mL MeCN. A 200 μL portion of stock solution wasadded to 200 μL of FLAG-angiogenin (2.9 mg/mL in 10 mM Bis-Tris buffer,pH 6.0). The resulting mixture was mutated for 12 hours at 25° C. Theextent of labeling was determined by MALDI-TOF mass spectrometry (datanot shown).

HeLa cell cells were grown to confluence in a 10-cm² dish beforecollection and lysis using M-PER protein extraction reagent from ThermoFisher Scientific. Esterase activity was verified by a colorimetricassay using p-nitrophenylacetate. 10 μL of FLAG-angiogenin labeled witheither diazo 3 or diazo 4 was added to 10 μL of cell lysate andincubated at 25° C. overnight. FLAG-angiogenin was re-isolated usingmagnetic Anti-FLAG M2 beads from Sigma-Aldrich. The removal of alllabels was confirmed using MALDI-TOF mass spectrometry (data not shown).

Example 8 Ultraviolet Spectra of Diazo Compound 2

The ultraviolet spectra of diazo compound 2 were measured over theconcentration range 0.8-50 mM, see FIG. 6A. A plot (FIG. 6B) of theconcentration dependence of the absorbance of diazo compound 2 (0.8-50mM) at λ_(max)=435 nm, gave ϵ=30.5 M⁻¹cm⁻¹.

Example 9 Summary of Results

Diazo compounds 1-6 were accessed from derivatives of phenylacetic acidFIG. 1B) as described in examples above. Briefly, an azide was installedat the benzylic position of the acid either through displacement of abromide or by diazo transfer to an existing amine. The ensuing α-azidoacids were then coupled to benzylamine and converted to the diazocompound by deimidogenation using a phosphinoester [5a,b].

In initial experiments, the effect of electron distribution on thereactivity of diazo groups was assessed by measuring the rate ofesterification in acetonitrile. Diazo compounds 1-6 were first reactedwith BocGlyOH, and the second-order rate constants were measured using¹H NMR spectroscopy. The effect of electron distribution on the reactionrate was dramatic: rate constants spanned over two orders of magnitudeand increased with the electron-donating character of the phenylsubstituents (FIG. 2A). Hammett analysis of these rate constants gave aslope of ρ=−2.7 (FIG. 2B). This value is comparable to those for typicalSN1 reactions and indicates that the esterification reaction is highlysensitive to substituents and that substantial positive chargeaccumulates during its course, [33] as expected from a mechanisminvolving an intermediate diazonium ion (Scheme 1, [27a, b]).

Next selectivity for esterification over hydrolysis in an aqueousenvironment was assessed. Towards that end, diazo compounds 1-6 werereacted with equimolar BocGlyOH in a 1:1 mixture of acetonitrile and2-(N-morpholino)ethanesulfonic acid (MES)-HCl buffer at pH 5.5, and wedetermined the ratio of ester-to-alcohol product with ¹H NMRspectroscopy. Surprisingly, the ester:alcohol ratio reached a maximum of1.4:1 and remained unchanged despite increasing electron-withdrawal bythe substituents (FIGS. 3A-B). This result is consistent with a sharpcutoff for the formation of a carboxylate-diazonium intimate ion- pairintermediate that is maintained in a solvent cage by a Coulombicinteraction (Scheme 1) [27a, b, 34].

Additional experiments were conducted with diazo compound 2 whichdemonstrated the fastest rate of those compounds that retainedchemoselectivity in an aqueous environment. Certain diazo compoundsundergo O—H and S—H insertion reactions [23c, 25a,b]. Diazo compound 2was assessed to determine if it would esterify acids selectively in thepresence of the sulfhydryl, hydroxyl, or phenolic moieties found onprotein side chains. Diazo compound 2 esterified BocSerO H,p-hydroxybenzoic acid, and 3-mercaptopropionic acid in 1:1acetonitrile/100 mM MES-HCl buffer at pH 5.5, and that no other couplingproducts were observable by ¹H NMR spectroscopy.

Additionally, the ability of diazo compound 2 for the labeling of aprotein was compared to that of 9-diazofluorene. The well-known modelprotein ribonuclease A [21] was treated with 10 equiv of each diazocompound. The reactions were allowed to proceed for 4 h at 37° C. in 1:1acetonitrile/10 mM MES-HCl buffer at pH 5.5. The extent ofesterification with both diazo reagents was determined using MALDI-TOFmass spectrometry. Diazo compound 2 was approximately twofold moreeffective than was 9 diazofluorene in effecting esterification (FIGS.5A-B). Representative diazo compound 2 can be used to esterify proteinsin an aqueous environment very efficiently.

Example 10 Preparation of α-Diazo NHS Ester

α-Azido-4-methylphenyl N-hydroxysuccinimidyl ester (7) was synthesizedas described above. This compound 7 (3.4 g, 11.6 mmol) was dissolved in20:3 THF/H₂O (50 mL). N-Succinimidyl 3-(diphenylphosphino)propionate(4.5 g, 12.8 mmol) was added under N₂(g), and the reaction mixture wasstirred for 5 h. Triethylamine (2.3 g, 23.2 mmol) was added, and thesolution was stirred for 1 h. The solution was diluted with brine (20mL) and extracted with CH₂Cl₂ (2×10 mL). The organic layer was driedover anhydrous Na₂SO₄(s) and concentrated under reduced pressure. Theresidue was purified by chromatography on silica gel, eluting with 3:7EtOAc/hexanes to afford α-diazo NHS ester 8 (0.31 g, 10%) as an orangesolid.

Data for α-diazo NHS ester: ¹H NMR (500 MHz, CDCl₃, δ): 7.32 (d, 2H,J=8.3 Hz), 7.22 (d, 2H, J=8.1 Hz), 2.88 (s, 4H), 2.35 (s, 3H). ¹³C NMR(125 MHz, CDCl₃, δ): 169.4, 160.5, 137.1,129.9, 124.6, 119.8, 25.6,21.08 HRMS (ASAP-MS) m/z calcd for C₁₃H₁₁N₃O₄ [M−N₂+H]⁺ 246.0761; found246.0764.

Compound 8 is an exemplary compound of formula II which can be used tosynthesize compounds of formula I.

Example 11 Preparation of Additional α-Diazo Acetamides

A. α-Azido-4-Methylphneyl-N-Propargylacetamide

α-Azido-4-methylphenyl N-hydroxysuccinimidyl ester 7(1.1 g, 3.7 mmol)was dissolved in CH₂Cl₂ (20 mL). Propargylamine (0.2 g, 4.0 mmol) wasadded, and the reaction mixture stirred overnight. The solution wasconcentrated under reduced pressure. The residue was dissolved in EtOAc,and washed twice with saturated aqueous NaHCO₃ (2×10 mL). The organiclayer was dried over anhydrous Na₂SO₄(s) and concentrated under reducedpressure to afford α-azido-4-methylphenyl-N-propargylacetamide 9 (0.6 g,75%) as an off-white solid.

Data for α-azido-4-methylphenyl-N-propargylacetamide: ¹H NMR (400 MHz,CDCl₃, δ): 7.25 (d, 2H, J=6.3 Hz), 7.21 (d, 2H, J=8.1 Hz), 6.64 (s, 1H),5.03 (s, 1H), 4.08 (dd, 2H, J=2.5 Hz, 5.25 Hz), 2.36 (s, 3H), 2.26 (t,1H, J=2.4 Hz). ¹³C NMR (125 MHz, CDCl₃, δ): 167.8, 139.3, 131.6, 129.8,127.7, 79.9, 72.1, 67.0, 29.4, 21.2. HRMS (ESI⁺) m/z calcd for C₁₂H₁₂N₄O[M+H]⁺ 229.1084; found 229.1085.

B. Preparation of α-Diazo-4-Methylphenyl-N-Propargylacetamide

α-Azido-4-methylphenyl-N-propargylacetamide (0.6 g, 2.7 mmol) wasdissolved in a solution of 20:3 THF/H₂O (16 mL). N-Succinimidyl3-(diphenylphosphino)propionate (1.1 g, 3.0 mmol) was added under N₂(g),and the reaction mixture was stirred for 5 h.1,8-Diazabicycloundec-7-ene (DBU; 0.8 g, 5.5 mmol) was added, and thesolution was stirred overnight. The solution was diluted with brine (20mL) and extracted with CH₂Cl₂ (2×10 mL). The organic layer was driedover anhydrous Na₂SO₄(s) and concentrated under reduced pressure. Theresidue was purified by chromatography on silica gel, eluting with 3:7EtOAc/hexanes to afford α-diazo-N-propargylacetamide (0.176 g, 30%) as ared solid.

Data for α-diazo-4-methylphenyl-N-propargylacetamide: ¹H NMR (500 MHz,CDCl₃, δ): 7.28-7.24 (m, 4H), 5.52 (s, 1H), 4.15-4.14 (dd, 2H, J=2.5,5.4 Hz), 2.38 (s, 3H), 2.23 (s, 1H). ¹³C NMR (125 MHz, CDCl₃, δ): 164.9,138.3, 130.5, 128.0, 122.6, 79.6, 71.6, 64.0, 29.7, 21.2. HRMS (ESI⁺)m/z calcd for C₁₂H₁₁N₃O [M+H]⁺ 214.0975; found 214.0975.

Example 12 Preparation of Compounds of Formula I Using Compounds ofFormula II:

A. α-Diazo-4-Methylphenyl-N-Methylacetamide

α-Diazo NHS ester 8 (100 mg, 0.37 mmol) was dissolved in CH₂Cl₂ (37 mL).Methylamine (0.2 mL of a 2.0 M solution in THF; 0.41 mmol) andN,N-diisopropylethylamine (DIEA; 143 mg, 1.1 mmol) were added, and thereaction mixture was stirred overnight. The solution was concentratedunder reduced pressure, and the residue was dissolved in EtOAc. Theresidue was purified by chromatography on silica gel, eluting with 3:7EtOAc/hexanes to afford α-diazo-4-methyphenyl-N-methylacetamide (34 mg,49%) as a red solid.

Data for α-diazo-4-methylphenyl-N-methylacetamide: ¹H NMR (500 MHz,CDCl₃, δ): 7.26-7.25 (m, 4H), 5.36 (s, 1H), 2.90 (d, 3H, J=4.8 Hz), 2.37(s, 3H). ¹³C NMR (125 MHz, CDCl₃, δ): 165.8, 138.0, 130.4, 127.9, 123.2,63.7, 27.0, 21.2. HRMS (ESI⁺) m/z calcd for C₁₀H₁₁N₄O [M−N₂+H]⁺162.0913; found 162.0915.

B. Preparation of α-Diazo-4-Methylphenyl-N,N-Dimethylacetamide

α-Diazo NHS ester 8 (100 mg, 0.37 mmol) was dissolved in CH₂Cl₂ (37 mL).Dimethylamine (0.2 mL of a 2.0 M solution in THF; 0.41 mmol) and DIEA(143 mg, 1.1 mmol) were added, and the reaction mixture was stirredovernight. The solution was concentrated under reduced pressure, and theresidue was dissolved in EtOAc. The residue was purified bychromatography on silica gel, eluting with 3:7 EtOAc/hexanes to affordα-diazo-4-methylphenyl-N,N-dimethyl acetamide (24 mg, 32%) as a redsolid.

Data for α-diazo-N,N-dimethylacetamide: ¹H NMR (500 MHz, CDCl₃, δ): 7.19(d, 2H, J=8.1 Hz), 7.11 (d, 2H, J=8.3 Hz), 2.95 (s, 6H), 2.34 (s, 3H).¹³C NMR (125 MHz, CDCl₃, δ): 166.1, 135.6, 129.9, 124.7, 124.4, 62.4,37.8, 21.0. HRMS (ESI⁺) m/z calcd for C₁₁H₁₃N₃O [M−N₂+H]⁺ 176.1070;found 176.1071.

C. Preparation of α-Diazo-4-Methylphenyl-N-Pentylacetamide

α-Diazo NHS ester 8 (100 mg, 0.37 mmol) was dissolved in CH₂Cl₂ (37 mL).Pentylamine (35.4 mg, 0.41 mmol) and DIEA (143 mg, 1.1 mmol) were added,and the reaction mixture was stirred overnight. The solution wasconcentrated under reduced pressure, and the residue was dissolved inEtOAc. The residue was purified by chromatography on silica gel, elutingwith 1:4 EtOAc/hexanes to affordα-diazo-4-methylphenyl-N-pentylacetamide (65 mg, 72%) as a red solid.

Data for α-Diazo-4-methylphenyl-N-pentylacetamide: ¹H NMR (500 MHz,CDCl₃, δ): 7.26-7.23 (m, 4H), 5.37 (s, 1H), 3.36-3.32 (q, 2H, J=7.0 Hz),2.38 (s, 3H), 1.53-1.49 (m, 2H), 1.33-1.28 (m, 4H), 0.90-0.88 (t, 3H,J=6.9 Hz). ¹³C NMR (125 MHz, CDCl₃, δ): 164.9, 137.9, 130.4, 127.8,123.3, 63.8, 40.2, 29.6, 29.0, 22.3, 21.2, 14.0. HRMS (ESI⁺) m/z calcdfor C₁₄H₁₉N₃O [M−N₂+H]⁺ 218.1539; found 218.1541.

Example 13 Esterification of Proteins and Internalization

GFP was esterified as described above with five exemplary diazocompounds 2, 10-13 (FIG. 7A). The GFP variant used in these experimentsand its production were described previously [24]. Using massspectrometry, an average of ˜3-11 labels per protein were found (FIG.7B). Less polar diazo compounds tended to provide more extensivelabeling.

Chinese hamster ovary (CHO) K1 cells were incubated at 37° C. for 2 h inF-12K medium (which was supplemented with penicillin/streptomycin)containing either unlabeled or labeled GFP (15 μM). Internalization ofGFP was then quantified with flow cytometry, counting only live, singlecells, as shown in FIG. 8. More extensively labeled GFPs tended to beinternalized more efficiently. Individual cells were imaged by confocalmicroscopy for two of the diazo compounds. Esterification with eitherdiazo compound 11 or 12 enhanced the uptake of GFP into CHO K1 cells, asshown in FIGS. 9A-C. The images shown demonstrate that the labelledproteins are inside of the cell.

These data indicate that protein internalization is enhanced by formingesters with the diazo compounds of the invention.

One barrier to cellular entry of a protein into a cell is the Coulombicrepulsion between negatively charged amino acid residues on the proteinand negatively charged cell membrane components. Without wishing to bebound by any particular theory it is presently believed based on theresults of FIGS. 8 and 9A-C that masking of negative charges on aprotein by esterification facilitates cell penetration.

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We claim:
 1. A compound of formula I:

or salts thereof, where: R_(A) represents 1 to 3 non-hydrogensubstituents on the phenyl ring, wherein the non-hydrogen substituentsare selected from the group consisting of alkyl, cycloalkyl, alkoxy,cycloalkoxy, aryl, arylalkyl, halogen, haloalkyl, haloalkoxy,heterocyclyl and Rp—CO—NH—, where the alkyl, cycloalkyl, alkoxy,cycloalkoxy, aryl, arylalkyl and heterocyclyl groups are optionallysubstituted with 1-3 non-hydrogen substituents selected from alkyl,alkoxy, halogen, haloalkyl or haloalkoxy groups and Rp is hydrogen, analkyl group or R_(M), wherein R_(A) includes a non-hydrogen group whichhas a Hammett σ_(p) (para-sigma) or σ_(m) (meta-sigma) value of −0.2 to+0.1; R is an alkyl, alkenyl, alkynyl group or hydrogen, and R_(M) is anon-polymeric organic group M, having from 1 to 100 carbon atoms andoptionally nitrogen, oxygen or sulfur atoms, or is —L—M, where —L— is adivalent linker moiety having from 1-30 carbon atoms and optionallynitrogen, oxygen or sulfur atoms; R_(M) is a polymer directly bonded tothe compound or bonded to the compound by the divalent linker —L—; or Rand R_(M), together with the nitrogen to which they are bonded, form anoptionally substituted 5- to 10-member ring system, which optionallycontains one or two heteroatoms in addition to the N.
 2. The compound ofclaim 1, wherein R_(M) is an alkyl, alkynyl, cycloalkyl, aryl,arylalkyl, heterocyclyl or heteroaryl group which is optionallysubstituted with one or more alkyl, alkoxy, aryl, alkylaryl, halogen,haloalkyl, or haloalkoxy groups.
 3. The compound of claim 1, whereinR_(M) an alkyl, alkenyl, alkynyl or aryl group.
 4. The compound of claim1, wherein R_(M) is a label, a cell penetrating group, a cell targetinggroup, or a reactive group or a latent reactive group.
 5. The compoundof claim 1, wherein R_(M) is a cell targeting group and the celltargeting group is a protein, a polypeptide, a peptide, an antibody or afunctional fragment of an antibody.
 6. The compound of claim 1, whereinR_(M) is a polymer directly bonded to the compound or bonded to thecompound by the divalent linker —L—.
 7. The compound of claim 1, whereinR_(M) is a hydrophilic polymer.
 8. The compound of claim 1, whereinR_(M) is biotin or a derivative thereof which is optionally indirectlybonded to the compound by the divalent linker —L—.
 9. The compound ofclaim 1, wherein R_(M) comprises an amine or a thiol reactive group. 10.The compound of claim 1, wherein R and R_(M), together with the nitrogento which they are bonded, form an optionally substituted 5- to 10-memberring system, which optionally contains one or two heteroatoms inaddition to the N.
 11. The compound of claim 10, wherein R and R_(M),together with the nitrogen to which they are bonded, form an optionallysubstituted piperazine.
 12. The compound of claim 1, wherein R_(A)represents ring substitution having at least one non-hydrogen group atthe para or meta ring position.
 13. The compound of claim 1, whereinR_(A) represents ring substitution of a non-hydrogen group at the paraor meta ring position.
 14. The compound of claim 1, wherein R_(A)represents ring substitution of a non-hydrogen group at the para ringposition.
 15. The compound of claim 1, wherein R_(A) represents ringsubstitution at the para or meta ring position by a group selected fromalkyl, cycloalkyl, alkoxy, cycloalkoxy, aryl, arylalkyl, halogen,haloalkyl, haloalkoxy, heterocyclyl and Rp—CO—NH—, where the alkyl,cycloalkyl, alkoxy, cycloalkoxy, aryl, arylalkyl and heterocyclyl groupsare optionally substituted with 1-3 non-hydrogen substituents selectedfrom alkyl, alkoxy, halogen, haloalkyl and haloalkoxy groups.
 16. Thecompound of claim 1, wherein R_(A) is a Rp—CO—NH— group at the para ormeta ring position, where Rp is M.
 17. The compound of claim 1, whereinR_(A) is p-methyl, and R is hydrogen.
 18. The compound of claim 1,wherein R_(A) is p-methoxyl, and R is hydrogen.
 19. A method foresterifying one or more carboxylic acid groups in an organic orbiological molecule which comprises contacting the organic or biologicalmolecule with a compound of claim
 1. 20. A method for targeting anorganic or biological molecule having one or more carboxylate groups toa cell by esterifying the one or more carboxylic acid group of themolecule by the method of claim
 18. 21. A method for enhancing cellularuptake of an organic or biological molecule having one or morecarboxylate groups by esterifying the one or more carboxylate group ofthe cargo molecule by the method of claim
 18. 22. A method for labellingone or more carboxylic acid groups in an organic or biological moleculewhich comprises contacting the organic or biological molecule with acompound of claim
 1. 23. The compound of formula:

or salts thereof, where: R_(A) represents 1 to 3 non-hydrogensubstituents on the phenyl ring, wherein the non-hydrogen substituentsare selected from the group consisting of alkyl, cycloalkyl, alkoxy,cycloalkoxy, aryl, arylalkyl, halogen, haloalkyl, haloalkoxy,heterocyclyl and Rp—CO—NH—, where the alkyl, cycloalkyl, alkoxy,cycloalkoxy, aryl, arylalkyl and heterocyclyl groups are optionallysubstituted with 1-3 non-hydrogen substituents selected from alkyl,alkoxy, halogen, haloalkyl or haloalkoxy groups and Rp is hydrogen, analkyl group or R_(M), wherein R_(A) includes a non-hydrogen group whichhas a Hammett σ_(p) (para-sigma) or σ_(m) (meta-sigma) value of −0.2 to+0.1; R_(M) is a non-polymeric organic group M, having from 1 to 100carbon atoms and optionally nitrogen, oxygen or sulfur atoms, or is—L—M, where —L— is a divalent linker moiety having from 1-30 carbonatoms and optionally nitrogen, oxygen or sulfur atoms; or R_(M) is apolymer directly bonded to the compound or bonded to the compound by thedivalent linker —L—; and AC is a leaving group of an activated ester.24. The compound of claim 23 of formula:

wherein E is hydrogen or a —SO₃-(sulfo) salt.
 25. The compound of claim24, wherein R_(A) is an alkyl or alkoxy at the para position of theindicated phenyl ring.